Methods and compositions for preserving the viability of photoreceptor cells

ABSTRACT

Provided are methods and compositions for maintaining the viability of photoreceptor cells following retinal detachment. The viability of photoreceptor cells can be preserved by administering a neuroprotective agent, for example, a substance capable of suppressing endogenous MCP-1, a MCP-1 antagonist, a substance capable of suppressing endogenous TNF-alpha, a TNF-alpha antagonist, a substance capable of suppressing endogenous IL-1 beta, an IL-1 beta antagonist, a substance capable of inducing endogenous bFGF, exogenous bFGF, a bFGF mimetic, and combinations thereof, to a mammal having an eye with retinal detachment. The neuroprotective agent maintains the viability of the photoreceptor cells until such time that the retina becomes reattached to the underlying retinal pigment epithelium and choroid. The treatment minimizes the loss of vision, which otherwise may occur as a result of retinal detachment.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to and the benefit of U.S. Ser.No. 60/564,717, filed on Apr. 23, 2004, the entirety of which isincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to methods and compositions forpreserving the viability of photoreceptor cells following retinaldetachment, and more particularly the invention relates to compositionsincluding, for example, a neuroprotective agent, and their use inmaintaining the viability of photoreceptor cells following retinaldetachment.

BACKGROUND

The retina is a delicate neural tissue lining the back of the eye thatconverts light stimuli into electric signals for processing by thebrain. Within the eye, the retina is disposed upon underlying retinalpigment epithelium and choroid, which provide the retina with a supplyof blood and nutrients. A common and potentially blinding conditionknown as retinal detachment occurs when the retina becomes disassociatedfrom its underlying retinal pigment epithelium and/or choroid with theaccumulation of fluid in the intervening space. The loss of visualfunction appears to be more pronounced when the retinal detachmentsinvolve the central macula.

Unless treated, retinal detachments often result in irreversible visualdysfunction, which can range from partial to complete blindness. Thevisual dysfunction is believed to result from the death of photoreceptorcells, which can occur during the period when the retina is detachedfrom its underlying blood and nutrient supply. Reattachment of theretina to the back surface of the eye typically is accomplishedsurgically, and despite the good anatomical results of these surgeries(i.e., reattachment of the retina) patients often are still left withpermanent visual dysfunction.

There is still a need for new methods and compositions for maintainingthe viability of photoreceptor cells following retinal detachment andfor preserving vision when the retina ultimately becomes reattached.

SUMMARY

Monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha(TNF-alpha), interleukin-1 beta (IL-1 beta), and/or basic fibroblastgrowth factor (bFGF) mRNA and/or protein expression levels are increasedin the retina following retinal detachment. Modulating the activity ofthese targets provides a neuroprotective effect in the retina. Thus,modulating MCP-1, TNF-alpha, IL-1 beta, and/or bFGF can maintain theviability of photoreceptor cells following retinal detachment andpreserve vision when the retina is reattached.

In one aspect, the invention provides a method of preserving theviability of photoreceptor cells disposed within a retina of a mammalianeye following retinal detachment. The method includes administering to amammal having an eye in which a region of the retina has been detachedan amount of a neuroprotective agent selected from a substance capableof suppressing endogenous MCP-1, a MCP-1 antagonist, a substance capableof suppressing endogenous TNF-alpha, a TNF-alpha antagonist, a substancecapable of suppressing endogenous IL-1 beta, an IL-1 beta antagonist, asubstance capable of inducing endogenous bFGF, exogenous bFGF, a bFGFmimetic, and combinations thereof sufficient to preserve the viabilityof photoreceptor cells disposed within the region of the detachedretina. Suppressing endogenous cytokines such as MCP-1, TNF-alpha orIL-1 beta includes, but is not limited to, suppressing or otherwiseinterfering with expression of the gene encoding the cytokine,suppressing or otherwise interfering with the transcription of the geneinto mRNA, and/or suppressing or otherwise interfering with thetranslation of the mRNA from the cytokine gene into a functionalprotein.

This aspect can have any of the following features. The neuroprotectiveagent can be administered to the mammal prior to reattachment of theregion of detached retina. The neuroprotective agent can be administeredto the mammal after reattachment of the region of detached retina. Theneuroprotective agent can be administered locally or systemically. Aplurality of neuroprotective agents can be administered to the mammal.At least one neuroprotective agent can be administered by intraocular,intravitreal, or transcleral administration. The neuroprotective agentcan reduce the number of photoreceptor cells in the region that diefollowing retinal detachment. The retinal detachment occurs as a resultof a retinal tear, retinoblastoma, melanoma, diabetic retinopathy,uveitis, choroidal neovascularization, retinal ischemia, pathologicmyopia, or trauma.

In another aspect, the invention provides a method of preserving theviability of photoreceptor cells in a mammalian eye following retinaldetachment. More particularly, the invention provides a method ofpreserving the viability of photoreceptor cells disposed within a regionof a retina that has become detached from its underlying retinal pigmentepithelium and/or choroid. The method comprises administering to amammal in need of such treatment an amount of a neuroprotective agentsufficient to preserve the viability of photoreceptor cells, forexample, rods and/or cones, disposed within the region of the detachedretina. Administration of the neuroprotective agent minimizes the lossof visual function resulting from the retinal detachment.

The neuroprotective agent reduces the number of photoreceptor cells inthe region of the retina that, without treatment, would die followingretinal detachment. It is understood that photoreceptor cells in theretina may die via a variety of cell death pathways, for example, viaapoptotic and necrotic cell death pathways. It has been found, however,that upon retinal detachment, the photoreceptor cells undergo apoptoticcell death in the detached portion of the retina. Furthermore, one ormore caspases, for example, caspase 3, caspase 7 and caspase 9,participate in the cascade of events leading to apoptotic cell death.Accordingly, neuroprotective agents useful in the practice of theinvention can include, for example, an apoptosis inhibitor, for example,a caspase inhibitor, for example, one or more of, a caspase 3 inhibitor,a caspase 7 inhibitor, and a caspase 9 inhibitor.

Because photoreceptors die as a result of retinal detachment,administration of neuroprotective agents minimize or reduce the loss ofphotoreceptor cell viability until such time the retina becomesreattached to the choroid and an adequate blood and nutrient supply isonce again restored. The neuroprotective agent minimizes the level ofphotoreceptor cell death, and maintains photoreceptor cell viabilityprior to reattachment of the detached region of the retina. Undercertain circumstances, however, it may be beneficial to administer theneuroprotective agent for a period of time after a retinal detachmenthas been detected and/or the retina surgically reattached. This periodof time may vary and can include, for example, a period of a week, twoweeks, three weeks, a month, three months, six months, nine months, ayear, and two years, after surgical reattachment.

The neuroprotective agent, for example, can be administered, eitheralone or in combination with a pharmaceutically acceptable carrier orexcipient, by one or more routes. For example, the neuroprotective agentmay be administered systemically, for example, via oral or parenteralroutes, for example, via intravascular, intramuscular or subcutaneousroutes. Alternatively, the neuroprotective agent may be administeredlocally, for example, via intraocular, intravitreal, intraorbital, ortranscleral routes. Furthermore, it is contemplated that a plurality ofneuroprotective agents, for example, a substance capable of suppressingendogenous MCP-1, a MCP-1 antagonist, a substance capable of suppressingendogenous TNF-alpha, a TNF-alpha antagonist, a substance capable ofsuppressing endogenous IL-1 beta, an IL-1 beta antagonist, a substancecapable of inducing endogenous bFGF, exogenous bFGF, a bFGF mimetic, oneor more caspase inhibitors, and combinations thereof, may beadministered to the mammal to maintain viability of the photoreceptorcells disposed within the detached portion of the retina.

It is contemplated that the practice of the invention will be helpful inmaintaining the viability of photoreceptor cells in retinal detachmentsirrespective of how the retinal detachments were caused. For example, itis contemplated that the practice of the method of the invention will behelpful in minimizing visual dysfunction resulting from retinaldetachments caused by one or more of the following: a retinal tear,retinoblastoma, melanoma, diabetic retinopathy, uveitis, choroidalneovascularization, retinal ischemia, pathologic myopia, and trauma.

The foregoing aspects and embodiments of the invention may be more fullyunderstood by reference to the following figures, detailed descriptionand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the invention may be more fully understoodby reference to the drawings described below in which:

FIG. 1 depicts a bar chart showing the ratio of cleaved caspase 3 topro-caspase 3 in densitometry units in detached retinas (hatched bars)and attached retinas (solid bars) at one, three and five days postretinal detachment;

FIG. 2 depicts a bar chart showing the ratio of cleaved caspase 9 topro-caspase 9 in densitometry units in detached retinas (hatched bars)and attached retinas (solid bars) at one, three and five days postretinal detachment;

FIG. 3 depicts a bar chart showing the level of caspase 7 indensitometry units in detached retinas (hatched bars) and attachedretinas (solid bars) at one, three and five days post retinaldetachment;

FIG. 4 depicts a bar chart showing the ratio of cleaved poly-ADPribose-polymerase (PARP) to pro-PARP in densitometry units in detachedretinas (hatched bars) and attached retinas (solid bars) at one, threeand five days post retinal detachment;

FIG. 5 depicts a bar chart showing the retinal mRNA expression ofvarious types of mRNA in detached retina versus non-detached retina;

FIG. 6 depicts a bar chart showing retinal mRNA expressional changes fornineteen genes in detached retina versus non-detached retina at 72 hourspost detachment;

FIG. 7 depicts a bar chart showing retinal mRNA expressional changes fornineteen genes in detached retina of the right eye, or oculus dexter(OD), versus non-detached retina of the left eye, or oculus sinister(OS), at 72 hours post detachment;

FIG. 8 depicts a bar chart showing mRNA expressional changes in theretinal pigment layer (RPE), the outer nuclear layer (ONL), the innernuclear layer (INL) and the ganglion cell layer (GCL) of the retina, foreach of four genes (bFGF, TNF-alpha, IL-1 beta, and MCP-1), in detachedretina versus non-detached retina at 72 hours post detachment;

FIGS. 9A-E depict four bar charts and a line graph showing mRNAexpressional changes at 1 hour (FIG. 9A), 3 hours (FIG. 9B), 6 hours(FIG. 9C), and 24 hours (FIG. 9D) post retinal detachment, and a timecourse (FIG. 9E) showing these changes for certain genes;

FIG. 10A depicts a plot showing cytokine levels, as determined by ELISA,for TNF-alpha, IL-1 beta, and MCP-1 in detached retinas from right eyes(RD+) and undetached retinas from left eyes (RD−), at 6 and 72 hourspost retinal detachment;

FIG. 10B depicts an alternative view of the data depicted in FIG. 10A,with the y-axis showing the ratio of the average ELISA results forTNF-alpha, IL-1 beta, and MCP-1 in detached retinas from right eyes (OD)versus undetached retinas from left eyes (OS), at 6 and 72 hours postretinal detachment;

FIG. 11A depicts a bar chart showing quantitative results of TdT-dUTPTerminal Nick End-Labeling (TUNEL) staining of detached retina 24 hoursafter both retinal detachment and subretinal administration ofTNF-alpha, IL-1 beta, MCP-1, or control;

FIG. 11B depicts an alternative view of the data depicted in FIG. 11A,with the y-axis showing TUNEL-positive cells per square millimeter ofdetached retina 24 hours after both retinal detachment and subretinaladministration of TNF-alpha, IL-1 beta, MCP-1, or control;

FIG. 12 depicts a bar chart showing quantitative results of TUNELstaining of cells in the ONL of rats with a detached retina 72 hoursafter retinal detachment and subsequent treatment with etanercept;

FIG. 13 depicts a bar chart showing quantitative results of TUNELstaining of cells in the ONL of rats with a detached retina 72 hoursafter retinal detachment and subsequent treatment with either normalgoat serum (NGS) or goat anti-TNF-alpha antibody (TNFa);

FIG. 14 depicts a bar chart showing quantitative results of TUNELstaining of cells in the ONL of detached retina of either knockout micelacking the MCP-1 gene (CCL2) or wild-type control mice, 72 hours afterretinal detachment;

FIG. 15 depicts a bar chart showing quantitative results of TUNELstaining of cells in the ONL of detached retina of either knockout micedeficient in TNF-alpha (TNF KO), knockout mice deficient in TNFReceptors 1A and 1B (TNFR KO), or wild-type control mice 72 hours afterretinal detachment; and

FIG. 16 depicts a bar chart showing retinal mRNA expressional levels indetached retina from rats treated with an intravitreal injection of 5.0μg pigment epithelium-derived factor (PEDF) versus detached retina fromrats treated with an intravitreal injection of PBS control.

DETAILED DESCRIPTION

During retinal detachment, the entire retina or a portion of the retinabecomes dissociated from the underlying retinal pigment epithelium andchoroid. As a result, the sensitive photoreceptor cells disposed in thedetached portion of the retina become deprived of their normal supply ofblood and nutrients. If untreated, the retina or more particularly thesensitive photoreceptor cells disposed within the retina die causingpartial or even complete blindness. Accordingly, there is an ongoingneed for methods and compositions that preserve the viability ofphotoreceptor cells following retinal detachment. If photoreceptor celldeath can be minimized during retinal detachment, the affectedphotoreceptors likely will survive once the retina is reattached to theunderlying retinal pigment epithelium and choroid, and thephotoreceptors regain their normal blood and nutrient supply.

Retinal detachment can occur for a variety of reasons. The most commonreason for retinal detachment involves retinal tears. Retinaldetachments, however, can also occur because of, for example,retinoblastomas and other ocular tumors (for example, angiomas,melanomas, and lymphomas), diabetic retinopathy, retinal vasculardiseases, uveitis, retinal ischemia and trauma. Furthermore, retinaldetachments can occur as a result of formation of choroidalneovascularizations secondary to, for example, the neovascular form ofage-related macular degeneration, pathologic myopia, and ocularhistoplasmosis syndrome. It is understood that the clinical pathologiesof retinal detachments are different from those of degenerative retinaldisorders, for example, retinitis pigmentosa and age-related maculardegeneration. However, the neuroprotective agents discussed herein maybe useful in treating retinal detachments that occur secondary to anunderlying degenerative retinal disorder. Accordingly, it iscontemplated that the methods and compositions of the invention may beuseful in minimizing or otherwise reducing photoreceptor cell deathfollowing retinal detachment, irrespective of the cause of thedetachment.

The invention provides a method of preserving the viability ofphotoreceptor cells in a mammalian, for example, a primate, for example,a human, eye following retinal detachment. More particularly, theinvention provides a method of preserving the viability of photoreceptorcells disposed within a region of a retina, which has become detachedfrom its underlying retinal pigment epithelium and/or choroid. Themethod may be particularly helpful in preventing vision loss when theregion of detachment includes at least a portion of the macula. Themethod comprises administering to a mammal in need of such treatment anamount of a neuroprotective agent sufficient to preserve the viabilityof photoreceptor cells disposed within the region of the detachedretina.

As used herein, the term “neuroprotective agent” means any agent that,when administered to a mammal, either alone or in combination with otheragents, minimizes or eliminates photoreceptor cell death (including bothnecrotic and apoptotic cell death) in a region of the retina that hasbecome detached from the underlying retinal pigment epithelium and/orchoroid. It is contemplated that useful neuroprotective agents include,for example, a substance capable of suppressing endogenous MCP-1, aMCP-1 antagonist, a substance capable of suppressing endogenousTNF-alpha, a TNF-alpha antagonist, a substance capable of suppressingendogenous IL-1 beta, an IL-1 beta antagonist, a substance capable ofinducing endogenous bFGF, exogenous bFGF, a bFGF mimetic, combinationsthereof, and apoptosis inhibitors, for example, caspase inhibitors, andcertain neurotrophic factors that prevent the onset or progression ofapoptosis. More specifically, useful neuroprotective agents may include,for example, a protein (for example, a growth factor, antibody or anantigen binding fragment thereof), a peptide (for example, an amino acidsequence less than about 25 amino acids in length, and optionally anamino acid sequence less that about 15 amino acids in length), a nucleicacid (for example, a deoxyribonucleic acid, ribonucleic acid, anantisense oligonucleotide, or an aptamer), a peptidyl nucleic acid (forexample, an antisense peptidyl nucleic acid), an organic molecule or aninorganic molecule, which upon administration minimizes photoreceptorcell death following retinal detachment. Additionally, interfering RNA(RNAi) techniques can be used. Neuroprotective agents alternatively oradditionally may protect against gliosis.

It is understood that photoreceptor cell death during retinaldetachments may occur as a result of either necrotic or apoptotic (alsoknown as programmed cell death) pathways. Both of these pathways arediscussed in detail in, for example, Kerr et al. (1972) BR. J. CANCER26: 239-257, Wyllie et al. (1980) INT. REV. CYTOLOGY 68: 251-306; Walkeret al. (1988) METH. ACHIE. EXP. PATHOL. 13: 18-54 and Oppenheim (1991)ANN. REV. NEUROSCI. 14: 453-501. Apoptosis involves the orderlybreakdown and packaging of cellular components and their subsequentremoval by surrounding structures (Afford & Randhawa (2000) J. CLIN.PATHOL. 53:55-63). In general, apoptosis, also referred to as anapoptotic pathway, does not result in the activation of an inflammatoryresponse. This is in contrast to necrotic cell death, which ischaracterized by the random breakdown of cells in the setting of aninflammatory response. Typically, during necrosis, also known as anecrotic pathway, a catastrophic event, for example, trauma,inflammation, ischemia or infection, typically causes uncontrolled deathof a large group of cells. There are a variety of assays available fordetermining whether cell death is occurring via a necrotic pathway or anapoptotic pathway (see, for example, Cook et al. (1995) INVEST.OPHTHALMOL. VIS. SCI. 36:990-996).

Apoptosis involves the activation of a genetically determined cellsuicide program that results in a morphologically distinct form of celldeath characterized by cell shrinkage, nuclear condensation, DNAfragmentation, membrane reorganization and blebbing (Kerr et al. (1972)BR. J. CANCER 26: 239-257). Assays for detecting the presence ofapoptotic pathways include measuring morphologic and biochemicalstigmata associated with cellular breakdown and packaging, such aspyknotic nuclei, apoptotic bodies (vesicles containing degraded cellcomponents) and internucleosomally cleaved DNA. This last feature isspecifically detected by binding and labeling the exposed 3′—OH groupsof the cleaved DNA with the enzyme terminal deoxynucleotidyl transferasein the staining procedure often referred to as the TdT-dUTP TerminalNick End-Labeling (TUNEL) staining procedure. It is believed that, atthe core of this process lies a conserved set of serine proteases,called caspases, which are activated specifically in apoptotic cells.

There are approximately fourteen known caspases, and the activation ofthese proteins results in the proteolytic digestion of the cell and itscontents. Each of the members of the caspase family possess anactive-site cysteine and cleave substrates at Asp-Xxx bonds (i.e., afterthe aspartic acid residue). In general, a caspase's substratespecificity typically is determined by the four residues amino-terminalto the cleavage site. Caspases have been subdivided into subfamiliesbased on their substrate specificity, extent of sequence identity andstructural similarities, and include, for example, caspase 1, caspase 2,caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8,caspase 9, caspase 10, caspase 11, caspase 12, caspase 13 and caspase14. Monitoring their activity can be used to assess the level ofon-going apoptosis.

Furthermore, it has been suggested that apoptosis is associated with thegeneration of reactive oxygen species, and that the product of the Bcl-₂gene protects cells against apoptosis by inhibiting the generation orthe action of the reactive oxygen species (Hockenbery et al. (1993) CELL75: 241-251, Kane et al. (1993) SCIENCE 262: 1274-1277, Veis et al.(1993) CELL 75: 229-240, Virgili et al. (1998) FREE RADICALS BIOL. MED.24: 93-101). Bcl-₂ belongs to a growing family of apoptosis regulatorygene products, which may either be death antagonists (Bcl-₂, Bcl-x_(L))or death agonists (Bax, Bak) (Kroemer et al. (1997) NAT. MED. 3:614-620). Control of cell death appears to be regulated by theseinteractions and by constitutive activities of the various familymembers (Hockenbery et al. (1993) CELL 75: 241-251). Several apoptoticpathways may coexist in mammalian cells that are preferentiallyactivated in a stimulus-, stage-, context-specific and cell-type manner(Hakem et al. (1998) CELL 94: 339-352). However, it is contemplated thatagents that upregulate the level of the Bcl-2 gene expression or slowdown the rate of breakdown of the Bcl-₂ gene product may be useful inthe practice of the invention.

Useful apoptosis inhibitors include, for example, (i) proteins, forexample, growth factors, cytokines, antibodies and antigen bindingfragments thereof (for example, Fab, Fab′, and Fv fragments),genetically engineered biosynthetic antibody binding sites, also knownin the art as BABS or sFv's, and (ii) peptides, for example, syntheticpeptides and derivatives thereof, which may be administered systemicallyor locally to the mammal. Other useful apoptosis inhibitors include, forexample, deoxyribonucleic acids (for example, antisenseoligonucleotides), ribonucleic acids (for example, antisenseoligonucleotides and aptamers) and peptidyl nucleic acids, which onceadministered reduce or eliminate expression of certain genes, forexample, caspase genes as in the case of anti-sense molecules, or canbind to and reduce or eliminate the activity of a target protein orreceptor as in the case of aptamers. Additionally, RNAi techniques canbe used. Other useful apoptosis inhibitors include small organic orinorganic molecules that reduce or eliminate apoptotic activity whenadministered to the mammal.

One set of apoptosis inhibitors useful in the practice of the inventioninclude caspase inhibitors. Caspase inhibitors include molecules thatinhibit or otherwise reduce the catalytic activity of a target caspasemolecule (for example, a classical competitive or non-competitiveinhibitor of catalytic activity) as well as molecules that prevent theonset or initiation of a caspase mediated apoptotic pathway.

With regard to the inhibitors of catalytic function, it is contemplatedthat useful caspase inhibitors include, on the one hand, broad spectruminhibitors that reduce or eliminate the activity of a plurality ofcaspases or, on the other hand, specific caspase inhibitors that reduceor eliminate the activity of a single caspase. In general, caspaseinhibitors act by binding the active site of a particular caspase enzymeand forming either a reversible or an irreversible linkage to targetcaspase molecule. Caspase inhibitors may include inhibitors of one ormore of caspase 1, caspase 2, caspase 3, caspase 4, caspase 6, caspase7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13,and caspase 14.

Useful caspase inhibitors include commercially available syntheticcaspase inhibitors. Synthetic caspase inhibitors typically include apeptide recognition sequence attached to a functional group such as analdehyde, chloromethylketone, fluoromethylketone, orfluoroacyloxymethylketone. Typically, synthetic caspase inhibitors withan aldehyde fu group reversibly bind to their target caspases, whereasthe caspase inhibitors with the otl functional groups tend to bindirreversibly to their targets. Useful caspase inhibitors, wl-modeledwith Michaelis-Menten kinetics, preferably have a dissociation constantof the enzyme-inhibitor complex (K_(i)) lower than 100 μM, preferablylower than 50 μM, more preferably lower than 1 μM. The peptiderecognition sequence corresponding to that found in endogenoussubstrates determines the specificity of a particular caspase. Forexample, peptides with the Ac-Tyr-Val-Ala-Asp-aldehyde sequence arepotent inhibitors of caspases 1 and 4 (K_(i)=10 nM), and are weakinhibitors of caspases 3 and 7 (K_(i)≧50 μM). Removal of the tyrosineresidue, however, results in a potent but less specific inhibitor. Forexample, 2-Val-Ala-Asp-fluoromethylketone inhibits caspases 1 and 4 aswell as caspases 3 and 7.

Exemplary synthetic caspase 1 inhibitors, include, for example,Ac-N-Me-Tyr-Val-Ala-Asp-aldehyde, Ac-Trp-Glu-His-Asp-aldehyde,Ac-Tyr-N-Me-Val-Ala-N-Me-Asp-aldehyde, Ac-Tyr-Val-Ala-Asp-Aldehyde,Ac-Tyr-Val-Ala-Asp-chloromethylketone,Ac-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethylketone,Ac-Tyr-Val-Ala-Asp(OtBu)-aldehyde-dimethyl acetol,Ac-Tyr-Val-Lys-Asp-aldehyde,Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone,biotinyl-Tyr-Val-Ala-Asp-chloromethylketone,Boc-Asp(OBzl)-chloromethylketone,ethoxycarbonyl-Ala-Tyr-Val-Ala-Asp-aldehyde (pseudo acid),Z-Asp-2,6-dichlorobenzoyloxymethylketone, Z-Asp(OlBu)-bromomethylketone,Z-Tyr-Val-Ala-Asp-chloromethylketone,Z-Tyr-Val-Ala-DL-Asp-fluoromethlyketone,Z-Val-Ala-DL-Asp-fluoromethylketone, andZ-Val-Ala-DL-Asp(OMe)-fluoromethylketone, all of which can be obtainedfrom Bachem Bioscience Inc., PA. Other exemplary caspase 1 inhibitorsinclude, for example, Z-Val-Ala-Asp-fiuoromethylketone,biotin-X-Val-Ala-Asp-fluoromethylketone, Ac-Val-Ala-Asp-aldehyde,Boc-Asp-fluoromethylketone,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Pro-Tyr-Val-Ala-Asp-aldehyde(SEQ ID NO: 1), biotin-Tyr-Val-Ala-Asp-fluoroacyloxymethylketone,Ac-Tyr-Val-Ala-Asp-acyloxymethylketone, Z-Asp-CH2-DCB,Z-Tyr-Val-Ala-Asp-fluoromethylketone, all of which can be obtained fromCalbiochem, Calif.

Exemplary synthetic caspase 2 inhibitors, include, for example,Ac-Val-Asp-Val-Ala-Asp-aldehyde, which can be obtained from BachemBioscience Inc., PA, and Z-Val-Asp-Val-Ala-Asp-fluoromethylketone, whichcan be obtained from Calbiochem, Calif.

Exemplary synthetic caspase 3 precursor protease inhibitors include, forexample, Ac-Glu-Ser-Met-Asp-aldehyde (pseudo acid) andAc-Ile-Glu-Thr-Asp-aldehyde (pseudo acid) which can be obtained fromBachem Bioscience Inc., PA. Exemplary synthetic caspase 3 inhibitorsinclude, for example, Ac-Asp-Glu-Val-Asp-aldehyde,Ac-Asp-Met-Gin-Asp-aldehyde, biotinyl-Asp-Glu-Val-Asp-aldehyde,Z-Asp-Glu-Val-Asp-chloromethylketone,Z-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fluoromethylketone, andZ-Val-Ala-DL-Asp(OMe)-fluoromethylketone which can be obtained fromBachem Bioscience Inc., PA. Other exemplary caspase 3 inhibitorsinclude, for example,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-aldehyde(SEQ ID NO: 2), Z-Asp-Glu-Val-Asp-fluoromethylketone,biotin-X-Asp-Glu-Val-Asp-fluoromethylketone,Ac-Asp-Glu-Val-Asp-chloromethylketone, which can be obtained fromCalbiochem, Calif.

Exemplary synthetic caspase 4 inhibitors include, for example,Ac-Leu-Glu-Val-Asp-aldehyde and Z-Tyr-Val-Ala-DL-Asp-fluoromethylketone,which can be obtained from Bachem Bioscience Inc., PA, andAc-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-Val-Pro-aldehyde(SEQ ID NO: 3), which can be obtained from Calbiochem, Calif.

Exemplary synthetic caspase 5 inhibitors include, for example,Z-Trp-His-Glu-Asp-fluoromethylketone, which can be obtained fromCalbiochem, Calif., and Ac-Trp-Glu-His-Asp-aldehyde andZ-Trp-Glu(O-Me)-His-Asp(O-Me) fluoromethylketone, which can be obtainedfrom Sigma Aldrich, Germany.

Exemplary synthetic caspase 6 inhibitors include, for example,Ac-Val-Glu-Ile-Asp-aldehyde, Z-Val-Glu-Ile-Asp-fluoromethylketone, andAc-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Glu-Ile-Asp-aldehyde(SEQ ID NO: 4), which can be obtained from Calbiochem, Calif.

Exemplary synthetic caspase 7 inhibitors include, for example,Z-Asp(OMe)-Gln-Met-Asp(OMe) fluoromethylketone,Ac-Asp-Glu-Val-Asp-aldehyde, Biotin-Asp-Glu-Val-Asp-fluoromethylketone,Z-Asp-Glu-Val-Asp-fluoromethylketone,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-aldehyde(SEQ ID NO: 2), which can be obtained from Sigma Aldrich, Germany.

Exemplary synthetic caspase 8 inhibitors include, for example,Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Ile-Glu-Pro-Asp-aldehyde,Ac-Ile-Glu-Thr-Asp-aldehyde, Ac-Trp-Glu-His-Asp-aldehyde andBoc-Ala-Glu-Val-Asp-aldehyde which can be obtained from BachemBioscience Inc., PA. Other exemplary caspase 8 inhibitors include, forexample,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile-Glu-Thr-Asp-aldehyde(SEQ ID NO: 5) and Z-Ile-Glu-Thr-Asp-fluoromethylketone, which can beobtained from Calbiochem, Calif.

Exemplary synthetic caspase 9 inhibitors, include, for example,Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Leu-Glu-His-Asp-aldehyde, andAc-Leu-Glu-His-Asp-chloromethylketone which can be obtained from BachemBioscience Inc., PA. Other exemplary caspase 9 inhibitors include, forexample, Z-Leu-Glu-His-Asp-fluoromethylketone andAc-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-His-Asp-aldehyde(SEQ ID NO: 6), which can be obtained from Calbiochem, Calif.

Furthermore, it is contemplated that caspase specific antibodies (forexample, monoclonal or polyclonal antibodies, or antigen bindingfragments thereof), for example, an antibody that specifically binds toand reduces the activity of, or inactivates a particular caspase may beuseful in the practice of the invention. For example, an anti-caspase 3antibody, an anti-caspase 7 antibody, or an anti-caspase 9 antibody maybe useful in the practice of the invention. Additionally, it iscontemplated that an anti-caspase aptamer that specifically binds andreduces the activity of, or inactivates a particular caspase, forexample, an anti-caspase 3 aptamer, an anti-caspase 7 aptamer, or ananti-caspase 9 aptamer may be useful in the practice of the invention.

Alternatively, endogenous caspase inhibitors can be used to reduce, orinhibit caspase activity. For example, one useful class of endogenouscaspase inhibitor includes proteins known as inhibitors of apoptosisproteins (IAPs) (Deveraux et al. (1998) EMBO JOURNAL 17(8): 2215-2223)including bioactive fragments and analogs thereof. One exemplary IAPincludes X-linked inhibitor of apoptosis protein (XIAP), which has beenshown to be a direct and selective inhibitor of caspase-3, caspase-7 andcaspase-9. Another exemplary IAP includes survivin (see, U.S. Pat. No.6,245,523; Papapetropoulos et al. (2000) J. BIOL. CHEM. 275: 9102-9105),including bioactive fragments and analogs thereof. Survivin has beenreported to inhibit caspase-3 and caspase-7 activity. It is alsocontemplated that molecules that act through IAPs will also be useful,for example, VEGF has anti-apoptotic activity by acting throughsurvivin.

In addition, it is contemplated that useful neuroprotective agents mayinclude one or more neurotrophic factors, which may serve as effectiveapoptosis inhibitors (Lewis et al. (1999) INVEST. OPHTHALMOL. VIS. SCI.40: 1530-44; LaVail et al. (1998) INVEST. OPHTHALMOL. VIS. SCI. 39:592-602). Exemplary neurotrophic factors include, for example, BrainDerived Growth Factor (Caffe et al. (2001) INVEST OPHTHALMOL. VIS. SCI.42: 275-82) including bioactive fragments and analogs thereof;Fibroblast Growth Factor (Bryckaert et al. (1999) ONCOGENE 18:7584-7593) including bioactive fragments and analogs thereof; PEDFincluding bioactive fragments and analogs thereof; and Insulin-likeGrowth Factors, for example, IGF-I and IGF-II (Rukenstein et al. (1991)J NEUROSCI. 11:2552-2563) including bioactive fragments and analogsthereof; and cytokine-associated neurotrophic factors.

Bioactive fragments refer to portions of an intact template protein thathave at least 30%, more preferably at least 70%, and most preferably atleast 90% of the biological activity of the intact proteins. Analogsrefer to species and allelic variants of the intact protein, or aminoacid replacements, insertions or deletions thereof that have at least30%, more preferably at least 70%, and most preferably 90% of thebiological activity of the intact protein.

With reference to the foregoing proteins, the term “analogs” includesvariant sequences that are at least 80% similar or 70% identical, morepreferably at least 90% similar or 80% identical, and most preferably95% similar or 90% identical to at least a portion of one of theexemplary proteins described herein, for example, Brain Derived GrowthFactor. To determine whether a candidate protein has the requisitepercentage similarity or identity to a reference polypeptide, thecandidate amino acid sequence and the reference amino acid sequence arefirst aligned using the dynamic programming algorithm described in Smithand Waterman (1981) J. MOL BIOL. 147:195-197, in combination with theBLOSUM62 substitution matrix described in FIG. 2 of Henikoff andHenikoff (1992), PROC. NAT. ACAD. SCI. USA 89:10915-10919. Anappropriate value for the gap insertion penalty is −12, and anappropriate value for the gap extension penalty is −4. Computer programsperforming alignments using the algorithm of Smith-Waterman and theBLOSUM62 matrix, such as the GCG program suite (Oxford Molecular Group,Oxford, England), are commercially available and widely used by thoseskilled in the art. Once the alignment between the candidate andreference sequence is made, a percent similarity score may becalculated. The individual amino acids of each sequence are comparedsequentially according to their similarity to each other. If the valuein the BLOSUM62 matrix corresponding to the two aligned amino acids iszero or a negative number, the pairwise similarity score is zero;otherwise the pairwise similarity score is 1.0. The raw similarity scoreis the sum of the pairwise similarity scores of the aligned amino acids.The raw score is then normalized by dividing it by the number of aminoacids in the smaller of the candidate or reference sequences. Thenormalized raw score is the percent similarity.

Alternatively, to calculate a percent identity, the aligned amino acidsof each sequence are again compared sequentially. If the amino acids arenon-identical, the pairwise identity score is zero; otherwise thepairwise identity score is 1.0. The raw identity score is the sum of theidentical aligned amino acids. The raw score is then normalized bydividing it by the number of amino acids in the smaller of the candidateor reference sequences. The normalized raw score is the percentidentity. Insertions and deletions are ignored for the purposes ofcalculating percent similarity and identity. Accordingly, gap penaltiesare not used in this calculation, although they are used in the initialalignment.

Furthermore, by way of example, cAMP elevating agents may also serve aseffective apoptosis inhibitors. Exemplary cAMP elevating agents include,for example, 8-(4-chlorophenylthio)-adenosine-3′:5′-cyclic-monophosphate(CPT-cAMP) (Koike (1992) PROG. NEURO-PSYCHOPHARMACOL. BIOL. PSYCHIAT.16: 95-106), forskolin, isobutyl methylxanthine, cholera toxin (Martinet al. (1992) J. NEUROBIOL. 23:1205-1220), and 8-bromo-cAMP, N⁶,O^(2′)-dibutyryl-cAMP and N⁶,O^(2′)dioctanoyl-cAMP (Rydel and Greene(1988) PROC. NAT. ACAD. SCI. USA 85: 1257-1261).

Furthermore, other exemplary apoptosis inhibitors can include, forexample, glutamate inhibitors, for example, NMDA receptor inhibitors(Bamford et al. (2000) EXP. CELL RES. 256: 1-11) such as eliprodil(Kapin et al. (1999) INVEST. OPHTHALMOL. VIS. SCI 40,1177-82) and MK-801(Solberg et al. INVEST. OPHTHALMOL. VIS. SCI (1997) 38,1380-1389) andn-acetylated-αππηα-linked-acidic dipeptidase inhibitors, such as,2-(phosphonomethyl) pentanedioic acid (2-PMPA) (Harada et al. NEUR.LETT. (2000) 292,134-36); steroids, for example, hydrocortisone anddexamethasone (see, U.S. Pat. No. 5,840,719; Wenzel et al. (2001)INVEST. OPHTHALMOL. VIS. SCI. 42: 1653-9); nitric oxide synthaseinhibitors (Donovan et al. (2001) J. BIOL. CHEM. 276: 23000-8); serineprotease inhibitors, for example, 3,4-dichloroisocoumarin andN-tosyl-lysine chloromethyl ketone (see, U.S. Pat. No. 6,180,402);cysteine protease inhibitors, for example, N-ethylmaleimide andiodoacetamide, or an interleukin-1 β-converting enzyme inhibitor, forexample, Z-Asp-2, 6-dichlorobenzoyloxymethylketone (see, U.S. Pat. No.6,180,402); and anti-sense nucleic acid or peptidyl nucleic acidsequences that lower of prevent the expression of one or more of thedeath agonists, for example, the products of the Bax, and Bak genes.

In addition, or in the alternative, it may be useful to inhibitexpression or activity of members of the caspase cascade that areupstream or downstream of caspase 3, caspase 7 and caspase 9. Forexample, it may be useful to inhibit PARP, which is a component of theapoptosis cascade downstream of caspase 7. An exemplary PARP inhibitorincludes 3-aminobenzamide (Weise et al. (2001) CELL DEATH DIFFER.8:801-807). Other examples include inhibitors of the expression oractivity of Apoptosis Activating Factor-1 (Apaf-1) and/or cytochrome C.Apaf-1 and cytochrome C bind the activated form of caspase 9 to producea complex, which is known to propagate the apoptosis cascade. Thus, anyprotein (for example, antibody), nucleic acid (for example, aptamer),peptidyl nucleic acid (for example, antisense molecule) or othermolecule that inhibits or interferes with the binding of caspase 9 toApaf-1/cytochrome C can serve to inhibit apoptosis.

Under certain circumstances, it may be advantageous to also administerto the individual undergoing treatment with the neuroprotective agent ananti-permeability agent and/or an inflammatory agent so as to minimizephotoreceptor cell death. An anti-permeability agent is a molecule thatreduces the permeability of normal blood vessels. Examples of suchmolecules include molecules that prevent or reduce the expression ofgenes encoding, for example, Vascular Endothelial Growth Factor (VEGF)or an Intercellular Adhesion Molecule (ICAM) (for example, ICAM-1,ICAM-2 or ICAM-3). Exemplary molecules include antisenseoligonucleotides and antisense peptidyl nucleic acids that hybridize invivo to a nucleic acid encoding a VEGF gene, an ICAM gene, or aregulatory element associated therewith. Other suitable molecules bindto and/or reduce the activity of, for example, the VEGF and ICAMmolecules (for example, anti-VEGF and anti-ICAM antibodies and antigenbinding fragments thereof, and anti-VEGF or anti-ICAM aptamers). Othersuitable molecules bind to and prevent ligand binding and/or activationof a cognate receptor, for example, the VEGF receptor or the ICAMreceptor. Such molecules may be administered to the individual in anamount sufficient to reduce the permeability of blood vessels in theeye. An anti-inflammatory agent is a molecule that prevents or reducesan inflammatory response in the eye and in some instances can beconsidered a neuroprotective agent. Exemplary anti-inflammatory agentsinclude steroids, for example, hydrocortisone, dexamethasone sodiumphosphate, and methylpredisolone. Such molecules may be administered tothe individual in an amount sufficient to reduce or eliminate aninflammatory response in the eye.

It is contemplated that the foregoing and other neuroprotective agentsnow known or hereafter discovered may be assayed for efficacy inminimizing photoreceptor cell death following retinal detachment using avariety of model systems. Basic techniques for inducing retinaldetachment in various animal models are known in the art (see, forexample, Anderson et al. (1983) INVEST. OPHTHALMOL. VIS. SCI. 24:906-926; Cook et al. (1995) INVEST. OPHTHALMOL. VIS. SCI. 36: 990-996;Marc et al. (1998) OPHTHALMOL. VIS. SCI. 39: 1694-1702; Mervin et al.(1999) AM. J. OPHTHALMOL. 128: 155-164; Lewis et al. (1999) AM. J.OPHTHALMOL. 128: 165-172). Once a suitable animal model has been created(see, Example 1 below) an established or putative neuroprotective agentcan be administered to an eye at different dosages. The ability of theneuroprotective agent and dosage required to maintain cell viability maybe assayed by one or more of (i) tissue histology, (ii) TUNEL staining,which quantifies the number of TUNEL-positive cells per section, (iii)electron microscopy, (iv) immunoelectron microscopy to detect the levelof, for example, apoptosis inducing factor (AIF) in the samples, and (v)immunochemical analyses, for example, via Western blotting, to detectthe level of certain caspases in a sample.

The TUNEL technique is particularly useful in observing the level ofapoptosis in photoreceptor cells. By observing the number ofTUNEL-positive cells in a sample, it is possible to determine whether aparticular neuroprotective agent is effective at minimizing or reducingthe level of apoptosis, or eliminating apoptosis in a sample. Forexample, the potency of the neuroprotective agent will have an inverserelationship to the number of TUNEL-positive cells per sample. Bycomparing the efficacy of a variety of potential neuroprotective agentsusing these methods, it is possible to identify neuroprotective factorsmost useful in the practice of the invention.

The neuroprotective agent may be administered to the mammal from thetime the retinal detachment is detected to the time the retina isrepaired, for example, via surgical reattachment. It is understood,however, that under certain circumstances, it may be advantageous toadminister the neuroprotective agent to the mammal even after the retinahas been surgically repaired. For example, even after the surgicalreattachment of a detached retina in patients with rhegmatogenousretinal detachments, persistent subretinal fluid may exist under thefovea as detected by ocular coherence tomography long after the surgeryhas been performed (see, Hagimura et al. (2002) AM. J. OPHTHALMOL.133:516-520). As a result, even after surgical repair the retina maystill not be completely reattached to the underlying retinal pigmentepithelium and choroid. Furthermore, when retinal detachments occursecondary to another disorder, for example, the neovascular form ofage-related macular degeneration and ocular melanomas, it may bebeneficial to administer the neuroprotective agent to the individualwhile the underlying disorder is being treated so as to minimize loss ofphotoreceptor cell viability. Accordingly, in such cases, it may beadvantageous to administer the neuroprotective agent to the mammal forone week, two weeks, three weeks, one month, three months, six months,nine months, one year, two years or more (i) after retinal detachmenthas been identified, and/or (ii) after surgical reattachment of theretina has occurred, and/or (iii) after detection of an underlyingdegenerative disorder, so as to minimize photoreceptor cell death.

Once the appropriate neuroprotective agents have been identified, theymay be administered to the mammal of interest in any one of a widevariety of ways. It is contemplated that a neuroprotective agent, forexample, a caspase inhibitor, can be administered either alone or incombination with another neuroprotective agent, for example, aneurotrophic agent. It is contemplated that the efficacy of thetreatment may be enhanced by administering two, three, four or moredifferent neuroprotective agents either together or one after the other.Although the best means of administering a particular neuroprotectiveagent or combination of neuroprotective agents may be determinedempirically, it is contemplated that neuroprotective agents may beadministered locally or systemically.

Systemic modes of administration include both oral and parenteralroutes. Parenteral routes include, for example, intravenous,intrarterial, intramuscular, intradermal, subcutaneous, intranasal andintraperitoneal routes. It is contemplated that the neuroprotectiveagents administered systemically may be modified or formulated to targetthe neuroprotective agent to the eye. Local modes of administrationinclude, for example, intraocular, intraorbital, subconjuctival,intravitreal, subretinal or transcleral routes. It is noted, however,that local routes of administration are preferred over systemic routesbecause significantly smaller amounts of the neuroprotective agent canexert an effect when administered locally (for example, intravitreally)versus when administered systemically (for example, intravenously).Furthermore, the local modes of administration can reduce or eliminatethe incidence of potentially toxic side effects that may occur whentherapeutically effective amounts of neuroprotective agent (i.e., anamount of a neuroprotective agent sufficient to reduce, minimize oreliminate the death of photoreceptor cells following retinal detachment)are administered systemically.

Administration may be provided as a periodic bolus (for example,intravenously or intravitreally) or as continuous infusion from aninternal reservoir (for example, from an implant disposed at an intra-or extra-ocular location (see, U.S. Pat. Nos. 5,443,505 and 5,766,242))or from an external reservoir (for example, from an intravenous bag).The neuroprotective agent may be administered locally, for example, bycontinuous release from a sustained release drug delivery deviceimmobilized to an inner wall of the eye or via targeted transscleralcontrolled release into the choroid (see, for example, PCT/US00/00207,PCT/US02/14279, Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI.41:1181-1185, and Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI.41:1186-1191). A variety of devices suitable for administering aneuroprotective agent locally to the inside of the eye are known in theart. See, for example, U.S. Pat. Nos. 6,251,090, 6,299,895, 6,416,777,6,413,540, and 6,375,972, and PCT/US00/28187.

The neuroprotective agent also may be administered in a pharmaceuticallyacceptable carrier or vehicle so that administration does not otherwiseadversely affect the recipient's electrolyte and/or volume balance. Thecarrier may comprise, for example, physiologic saline or other buffersystem.

In addition, it is contemplated that the neuroprotective agent may beformulated so as to permit release of the neuroprotective agent over aprolonged period of time. A release system can include a matrix of abiodegradable material or a material which releases the incorporatedneuroprotective agent by diffusion. The neuroprotective agent can behomogeneously or heterogeneously distributed within the release system.A variety of release systems may be useful in the practice of theinvention, however, the choice of the appropriate system will dependupon rate of release required by a particular drug regime. Bothnon-degradable and degradable release systems can be used. Suitablerelease systems include polymers and polymeric matrices, non-polymericmatrices, or inorganic and organic excipients and diluents such as, butnot limited to, calcium carbonate and sugar (for example, trehalose).Release systems may be natural or synthetic. However, synthetic releasesystems are preferred because generally they are more reliable, morereproducible and produce more defined release profiles. The releasesystem material can be selected so that neuroprotective agents havingdifferent molecular weights are released by diffusion through ordegradation of the material.

Representative synthetic, biodegradable polymers include, for example:polyamides such as poly(amino acids) and poly(peptides); polyesters suchas poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolicacid), and poly(caprolactone); poly(anhydrides); polyorthoesters;polycarbonates; and chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), copolymers and mixtures thereof.Representative synthetic, non-degradable polymers include, for example:polyethers such as poly(ethylene oxide), poly(ethylene glycol), andpoly(tetramethylene oxide); vinyl polymers-polyacrylates andpolymethacrylates such as methyl, ethyl, other alkyl, hydroxyethylmethacrylate, acrylic and methacrylic acids, and others such aspolyvinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate);poly(urethanes); cellulose and its derivatives such as alkyl,hydroxyalkyl, ethers, esters, nitrocellulose, and various celluloseacetates; polysiloxanes; and any chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), copolymers and mixtures thereof.

One of the primary vehicles currently being developed for the deliveryof ocular pharmacological agents is the poly(lactide-co-glycolide)microsphere for intraocular injection. The microspheres are composed ofa polymer of lactic acid and glycolic acid, which are structured to formhollow spheres. These spheres can be approximately 15-30 μm in diameterand can be loaded with a variety of compounds varying in size fromsimple molecules to high molecular weight proteins such as antibodies.The biocompatibility of these microspheres is well established (see,Sintzel et al. (1996) EUR. J. PHARM. BIOPHARM. 42: 358-372), andmicrospheres have been used to deliver a wide variety of pharmacologicalagents in numerous biological systems. After injection,poly(lactide-co-glycolide) microspheres are hydrolyzed by thesurrounding tissues, which cause the release of the contents of themicrospheres (Zhu et al. (2000) NAT. BIOTECH. 18: 52-57). As will beappreciated, the in vivo half-life of a microsphere can be adjusteddepending on the specific needs of the system.

The type and amount of neuroprotective agent administered may dependupon various factors including, for example, the age, weight, gender,and health of the individual to be treated, as well as the type and/orseverity of the retinal detachment to be treated. As with the modes ofadministration, it is contemplated, that the optimal neuroprotectiveagents and dosages of those neuroprotective agents may be determinedempirically. The neuroprotective agent preferably is administered in anamount and for a time sufficient to permit the survival of at least 25%,more preferably at least 50%, and most preferably at least 75%, of thephotoreceptor cells in the detached region of the retina.

By way of example, protein-, peptide- or nucleic acid-basedneuroprotective agents can be administered at doses ranging, forexample, from about 0.001 to about 500 mg/kg, optionally from about 0.01to about 250 mg/kg, and optionally from about 0.1 to about 100 mg/kg.Nucleic acid-based neuroprotective agents may be administered at dosesranging from about 1 to about 20 mg/kg daily. Furthermore, antibodiesthat are neuroprotective agents may be administered intravenously atdoses ranging from about 0.1 to about 5 mg/kg once every two to fourweeks. With regard to intravitreal administration, the neuroprotectiveagents, for example, antibodies, may be administered periodically asboluses in dosages ranging from about 10 μg to about 5 mg/eye, andoptionally from about 100 μg to about 2 mg/eye. With regard totranscleral administration, the neuroprotective agents may beadministered periodically as boluses in dosages ranging from about 0.1μg to about 1 mg/eye, and optionally from about 0.5 μg to about 0.5mg/eye.

The present invention, therefore, includes the use of a neuroprotectiveagent, for example, a substance capable of suppressing endogenous MCP-1,a MCP-1 antagonist, a substance capable of suppressing endogenousTNF-alpha, a TNF-alpha antagonist, a substance capable of suppressingendogenous IL-1 beta, an IL-1 beta antagonist, a substance capable ofinducing endogenous bFGF, exogenous bFGF, a bFGF mimetic, a caspaseinhibitor, and combinations thereof, in the preparation of a medicamentfor treating an ocular condition associated with a retinal detachment,for example, a loss of vision as a result of photoreceptor cell death inthe region of retinal detachment. A composition comprising one or moreneuroprotective agents, one agent optionally being a caspase inhibitor,may be provided for use in the present invention. The neuroprotectiveagent or agents may be provided in a kit which optionally may comprise apackage insert with instructions for how to treat the patient with theretinal detachment. For each administration, the neuroprotective agentmay be provided in unit-dosage or multiple-dosage form. Preferreddosages of the neuroprotective agents, however, are as described above.

It has also been observed, as more fully described in Examples 3 and 4below, that mRNA levels of bFGF, TNF-alpha, and IL-1 beta, and proteinlevels of bFGF, TNF-alpha, and IL-1 beta increase in retinas in responseto detachment of the retina from the underlying choroidal tissue. bFGFis a cytokine that has anti-inflammatory activity, while TNF-alpha andIL-1 beta are cytokines with pro-inflammatory activity. Accordingly, tothe extent the viability of photoreceptor cells disposed within a retina(as well as other cells disposed within the retina) is to be preserved,steps may be taken to exploit these natural biological responses byeither enhancing the anti-inflammatory substance or suppressing thepro-inflammatory substance.

For example, insofar as bFGF has anti-inflammatory activity, it ispossible to enhance the level of bFGF by either further inducing itsproduction or exogenously adding it, in order to provide furtheranti-inflammatory activity. Similarly, it is possible to add exogenousmolecules that mimic the activity of bFGF (a bFGF mimetic). Any of theseroutes may preserve the viability of photoreceptor cells disposed withina retina. Examples of the neuroprotective agents that can enhance thelevel of bFGF, supplement the level of bFGF, or mimic the activity ofbFGF, include proteins or peptides that are inducers of the bFGF gene,exogenous bFGF itself (whether isolated from a natural source ormanufactured using recombinant DNA techniques), peptides from the activeportion of the full size bFGF protein, and small molecules. In someinstances, one or more of the following may be useful to modulate bFGF:growth hormone (increases bFGF-mRNA), TGF-beta 1 (upregulates bFGF-mRNAexpression and bFGF levels), cell-associated heparin-like molecules andheparan sulfate proteoglycans (controls bioavailability of bFGF toocular cells), prostaglandin E2 (stimulates bFGF-mRNA expression),prolactin (stimulates bFGF-mRNA expression), nicotine (regulates bFGFproduction and increases bFGF-mRNA), CS23 (acid-stable mutein of bFGFwhich up-regulates bFGF-mRNA expression), forskolin and PMA (increasesbFGF-mRNA expression), acidosis (enhances bFGF-mRNA expression as wellas bFGF secretion), NYAG (Naoyi'an granule, may enhance bFGF expressionand suppress TNF), huangpi when paired with TGF-beta 1 (may increasebFGF), lansoprazole (increases bFGF levels), Silicone-containing geldressing (Silastic (SGS) and ClearSite) (increases levels of bFGF),angiotensin II (increases bFGF expression), Luteinizing hormone(increases bFGF expression), and finasteride (decreases bFGF levels—tothe extent bFGF decrease is desired). It should be understood that anyof the dosage strategies, drug formulations, or administration schedulesdescribed above are applicable to all of these neuroprotective agents.

Conversely, insofar as TNF-alpha and IL-1 beta have pro-inflammatoryactivity, one may decrease the level of one or both of these cytokinesor otherwise antagonize one or both of their activities, in order toreduce the pro-inflammatory activity. Examples of the neuroprotectiveagents that can suppress the level of TNF-alpha or antagonize theactivity of TNF-alpha, include, for example, etanercept (Example 5,below), estrogen (decreases TNF gene expression), inhibition of p38 MAPK(decreases TNF-alpha expression), chronic garlic administration(decreases TNF-alpha expression), eicosapentaenoic acid (decreasesTNF-alpha expression), and TBP2 and TBP3 (decrease TNF-alpha activity).Neuroprotective agents can also include growth factors, cytokines,antibodies, for example TNF-alpha blocking antibody as described inExample 6, below, and Di62-anti-TNF-alpha monoclonal antibody(decreases. TNF-alpha activity), and antigen binding fragments thereof(for example, Fab, Fab′, and Fv fragments), genetically engineeredbiosynthetic antibody binding sites, also known in the art as BABS orsFv's, and peptides, for example, synthetic peptides and derivativesthereof, which may be administered to systemically or locally to themammal. Other useful neuroprotective agents include, for example,deoxyribonucleic acids (for example, antisense oligonucleotides),ribonucleic acids (for example, antisense oligonucleotides, aptamers,and interfering RNA) and peptidyl nucleic acids, which once administeredreduce or eliminate expression of certain genes or can bind to andreduce or eliminate the activity of a target protein or receptor as inthe case of aptamers. Other useful neuroprotective agents include smallorganic or inorganic molecules that reduce or eliminate activity whenadministered to the mammal. Any of these routes may preserve theviability of photoreceptor cells disposed within a retina. It should beunderstood that any of the dosage strategies, drug formulations, oradministration schedules described above are applicable to all of theseneuroprotective agents.

Examples of the neuroprotective agents that can suppress the level ofIL-1 beta or antagonize the activity of IL-1 beta, include pseudo-ICEand ICEBERG (block IL-1 beta secretion), polymorphonuclear cell (PMN)inhibitors (decrease IL-1), glucocorticoids (decrease IL-1 betaexpression), cyclosporine combined with a steroid (decreases IL-1expression), 15-deoxy-Delta(12,14)-PGJ2 (PGJ2, decreases IL-1 beta andTNF-alpha expression and increases IL-1rn expression), PPARgamma ligands(decrease IL-1 secretion), cPKC and nPKC (decrease IL-1 beta andTNF-alpha production), IL-1rn (IL-1 antagonist), PMA (increases IL-1rnexpression), IL-10 (increases IL-1rn), retinoic acid (upregulates IL-1beta—to the extent an increase in IL-1 is desired), phorbol esters(increase IL-1 beta expression, —to the extent an increase in IL-1 isdesired), IFNs (increase or decrease IL-1/IL-1rn), andlipopolysaccharide (increases IL-1rn expression and increases IL-1).Neuroprotective agents also include growth factors, cytokines,antibodies and antigen binding fragments thereof (for example, Fab,Fab′, and Fv fragments), genetically engineered biosynthetic antibodybinding sites, also known in the art as BABS or sFv's, and (ii)peptides, for example, synthetic peptides and derivatives thereof, whichmay be administered to systemically or locally to the mammal. Otheruseful neuroprotective agents include, for example, deoxyribonucleicacids (for example, antisense oligonucleotides), ribonucleic acids (forexample, antisense oligonucleotides, aptamers, and interfering RNA) andpeptidyl nucleic acids, which once administered reduce or eliminateexpression of certain genes or can bind to and reduce or eliminate theactivity of a target protein or receptor as in the case of aptamers.Other useful neuroprotective agents include small organic or inorganicmolecules that reduce or eliminate activity when administered to themammal. Any of these routes may preserve the viability of photoreceptorcells disposed within a retina. It should be understood that any of thedosage strategies, drug formulations, or administration schedulesdescribed above are applicable to all of these neuroprotective agents.

One or more of these cytokines can be modulated to preserve theviability of photoreceptor cells disposed within a retina (as well asother cells disposed within the retina). It should be understood thatany of the dosage strategies, drug formulations, or administrationschedules described above are applicable to all of these neuroprotectiveagents. Additionally, while treatments involving these cytokineresponses in detached retinas above are described with relation to theiranti- or pro-inflammatory activity and potential for inducing celldeath, to the extent another mechanism is involved (for example, theability of these cytokines to affect a undesirable proliferative changein photoreceptor cells or apoptosis), similar strategies can be used tochoose neuroprotective agents that modulate the cytokines.

In addition, as shown in Examples 3 and 4, MCP-1 mRNA and MCP-1 proteinare increased in detached retinas as compared to non-detached retinas.The increase in this factor can induce migration of microglia andmacrophages to the detachment area for phagocytosis of the debrisproduced by apoptotic photoreceptors. These monocytes also may berelated to secretion of TNF-alpha, and further destruction ofphotoreceptors. Immunohistochemistry data indicates that MCP-1 (andbFGF) was increased in Muller cells three days after retinal detachment,and microglia migrate toward the Muller cells, which increase the MCP-1protein. Insofar as microglia and macrophages may be secondarily toxicto photoreceptors, suppression of the increase of MCP-1 followingretinal detachment may be beneficial.

Examples of the neuroprotective agents that can suppress the level ofMCP-1 or antagonize the activity of MCP-1, include, for example, ADR7and ADR22 (MCP-1 antagonists), renin-angiotensin system (RAS) inhibitors(decreases MCP-1 expression), naked plasmid encoding 7ND (MCP-1antagonist), dilazep (inhibits MCP-1 mRNA expression), fenofibric acid(inhibits MCP-1 mRNA expression), cetirizine (decreases MCP-1 levels),tenidap (decreases MCP-1 expression), dexamethasone (decreases MCP-1mRNA expression), IFN-gamma (inhibits lipopolysaccharide-inducibleMCP-1), blockers of PTK and PKC which are needed for MCP-1 geneexpression in human monocytes, triple helix-forming oligonucleotides(TFO's) (selective inhibitor of MCP-1 gene expression), LY294002(inhibits MCP-1 expression), olmesartan (inhibits MCP-1 and TNFexpression), suppressors of NF-kappaB (inhibits MCP-1 expression),wogonin (inhibits MCP-1 expression), and Platelet-activating factor(PAF) (stimulates MCP-1 expression—to the extent an increase in IL-1 isdesired). Neuroprotective agents can also include growth factors,cytokines, antibodies and antigen binding fragments thereof (forexample, Fab, Fab′, and Fv fragments), genetically engineeredbiosynthetic antibody binding sites, also known in the art as BABS orsFv's, and (ii) peptides, for example, synthetic peptides andderivatives thereof, which may be administered to systemically orlocally to the mammal. Other useful neuroprotective agents include, forexample, deoxyribonucleic acids (for example, antisenseoligonucleotides), ribonucleic acids (for example, antisenseoligonucleotides, aptamers, and interfering RNA) and peptidyl nucleicacids, which once administered reduce or eliminate expression of certaingenes or can bind to and reduce or eliminate the activity of a targetprotein or receptor as in the case of aptamers. Other usefulneuroprotective agents include small organic or inorganic molecules thatreduce or eliminate activity when administered to the mammal. Any ofthese routes may preserve the viability of photoreceptor cells disposedwithin a retina. These neuroprotective agents can be used in combinationwith the neuroprotective agents described above, for example with theneuroprotective agents related to the cytokines described above. Itshould be understood that any of the dosage strategies, drugformulations, or administration schedules described above are applicableto all of these neuroprotective agents.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present invention also consistessentially of, or consist of, the recited components, and that theprocesses of the present invention also consist essentially of, orconsist of, the recited processing steps. Further, it should beunderstood that the order of steps or order for performing certainactions are immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

In light of the foregoing description, the specific non-limitingexamples presented below are for illustrative purposes and not intendedto limit the scope of the invention in any way.

EXAMPLES Example 1: Detection of Caspase Activity Following RetinalDetachment

This example demonstrates that certain caspases, particularly caspases3, 7 and 9, are activated in photoreceptor cells following retinaldetachment.

Experimental retinal detachments were created using modifications ofpreviously published protocols (Cook et al. (1995) INVEST. OPHTHALMOL.VIS. SCI. 36(6):990-6; Hisatomi et al. (2001) AM. J. PATH.158(4):1271-8). Briefly, rats were anesthetized using a 50:50 mixture ofketamine (100 mg/ml) and xylazine (20 mg/ml). Pupils were dilated usinga topically applied mixture of phenylephrine (5.0%) and tropicamide(0.8%). A 20 gauge micro-vitreoretinal blade was used to create asclerotomy approximately 2 mm posterior to the limbus. Care was takennot to damage the lens during the sclerotomy procedure. A Glasersubretinal injector (20 gauge shaft with a 32 gauge tip,Becton-Dickinson, Franklin Lakes, N.J.) connected to a syringe filledwith 10 mg/ml of Healon® sodium hyaluronate (Pharmacia and UpjohnCompany, Kalamazoo, Mich.) then was introduced into the vitreous cavity.The tip of the subretinal injector was used to create a retinotomy inthe peripheral retina, and then the sodium hyaluronate was slowlyinjected into the subretinal space to elevate the retina from theunderlying retinal pigment epithelium. Retinal detachments were createdonly in the left eye (OS) of each animal, with the right eye (OD)serving as the control. In each experimental eye, approximately one halfof the retina was detached, allowing the attached portion to serve as afurther control.

Following creation of the experimental retinal detachment, intraocularpressures were measured before and immediately after retinal detachmentwith a Tono-pen. No differences in intraocular pressures were noted. Theretinal break created by the subretinal injector was confined only tothe site of the injection.

Light microscopic analysis of the detached retinas showed an increase inmorphologic stigmata of apoptosis as a function of time afterdetachment. Eyes then were enucleated one, three, five and seven daysafter creation of the retinal detachment. For light microscopicanalysis, the cornea and lens were removed and the remaining eyecupplaced in a fixative containing 2.5% glutaraldehyde and 2% formaldehydein 0.1M cacodylate buffer (pH 7.4) and stored at 4° C. overnight. Tissuesamples then were post-fixed in 2% osmium tetroxide, dehydrated ingraded ethanol, and embedded in epoxy resin. One-micron sections werestained with 0.5% toluidine blue in 0.1% borate buffer and examined witha Zeiss photomicroscope (Axiophot, Oberkochen, Germany).

At one day after creation of the detachment, pyknosis in the ONL wasconfined to the area of the peripheral retinotomy site through which thesubretinal injector was introduced. By three days, however, pyknoticnuclei were seen in the whole ONL of the retina in the area of thedetachment. Extrusion of pyknotic nuclei from the ONL into thesubretinal space was observed. The remaining layers of the retinaappeared morphologically normal. No inflammatory cells were seen, andthere was no apparent disruption of the retinal vasculature. Similarchanges were seen in sections from retinas detached for up to one week.No pyknotic nuclei were seen in the area of the attached retina or inthe fellow, non-detached eye. The amount of ONL pyknosis was similarbetween detachments of three-day or one week duration.

Disruption of the photoreceptor outer segments was a prominent featurein the detached retinas. Outer segments of the control eyes and theattached portions of the experimental eyes had an orderly, parallelarrangement. Detachments produced artifactually during tissue processingin these eyes did not alter the photoreceptor morphology. In contrast,the photoreceptor outer segments of detached retinas were severelydisorganized and lost their normal structural organization.Additionally, outer segments in attached areas had similar lengths,whereas the outer segments in detached areas showed variable lengths.

Internucleosomal DNA cleavage in photoreceptor cells was detected viaTUNEL staining. For TUNEL staining, the cornea and lens were not removedafter enucleation, but rather the whole eye was fixated overnight at 4°C. in a phosphate buffered saline solution of 4% paraformaldehydesolution (pH 7.4). Then, a section was removed from the superior aspectof the globe and the remaining eyecup embedded in paraffin and sectionedat a thickness of 6 μm. TUNEL staining was performed on these sectionsusing the TdT-Fragel DNA Fragmentation Detection Kit (Oncogene Sciences,Boston, Mass.) in accordance with the manufacturer's instructions.Reaction signals were amplified using a preformed avidin:biotinylated-enzyme complex (ABC-kit, Vector Laboratories, Burlingame,Calif.). Internucleosomally cleaved DNA fragments were stained withdiaminobenzidine (DAB) (staining indicates TUNEL-positive cells) andsections were then counterstained with methylene green.

TUNEL-positive cells were detected at all time points tested (one,three, five and seven days post-detachment). TUNEL-positive staining wasconfined only to the photoreceptor cell layer. Two eyes with retinaldetachments that persisted for two months were monitored. The TUNELassay at two months did not reveal any staining indicating the presenceof internucleosomally cleaved DNA. The prolonged detachment wasassociated with a marked reduction in the thickness of and number ofcell bodies contained in the ONL as compared to the non-detached retina.

Antibodies specific for caspases 3, 7, 9 and PARP were used in Westernblots to probe total retinal protein extracts at various times aftercreation of the retinal detachment. For Western blot analysis, retinasfrom both experimental and control eyes were manually separated from theunderlying retinal pigment epithelium/choroid at days one, three andfive after creation of the retinal detachment. In eyes with retinaldetachments, the experimentally detached portion of the retina wasseparated from the attached portion of the retina and analyzedseparately. Retinas were homogenized and lysed with buffer containing 1mM ethylene diaminetetraacetic acid/ethylene glycol-bis(2-aminoethylethel-N,N,N′,N′-tetraacetic acid/dithiothreitol, 10 mMHEPES pH 7.6, 0.5% (octylphenoxy)polyethoxyethanol (IGEPAL), 42 mMpotassium chloride, 5 mM magnesium chloride, 1 mM phenylmethanesulfonylfluoride and 1 tablet of protease inhibitors per 10 ml buffer (CompleteMini, Roche Diagnostics GmbH, Mannheim, Germany). Samples were incubatedfor 15 minutes on ice, and then centrifuged at 21,000 rpm at 4° C. for30 minutes. The protein concentration of the supernatant was determinedusing the Bio-Rad D_(C) Protein Assay reagents (Bio-Rad Laboratories,Hercules, Calif.).

Proteins were separated via sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (7.5% and 15% Tris-HCL Ready-Gels, Bio-RadLaboratories), in which 30 μg of total retinal protein were applied ineach lane. The fractionated proteins were transferred to a PVDF membrane(Immobilon-P, Millipore, Bedford, Mass.). The resulting membrane wasblocked with 5% non-fat dry milk in 0.1% TBST IGEPAL. The blockedmembranes then were incubated with antibodies against caspase 7(1:1,000; Cell Signaling Technology, Beverly, Mass.), caspase 9(1:1,000; Medical & Biological Laboratories, Naka-ku Nagoya, Japan),cleaved-caspase 3 (1:1,000; Cell Signaling Technology, Beverly, Mass.),caspase 3 (1:2000; Santa Cruz, Santa Cruz, Calif.) or PARP (1:1000; CellSignaling Technologies, Beverly, Mass.) overnight at 4° C. Bands weredetected using the ECL-Plus reagent (Amersham, Pharmacia, Piscataway,N.J.). Membranes were exposed to HyperFilm (Amersham) and densitometrywas preformed using ImageQuant 1.2 software (Molecular Dynamics, Inc.,Sunnyvale, Calif.). For each eye tested, densitometry levels werenormalized by calculating the ratio of the cleaved-form to the pro-formof the protein of interest. Pro-caspase 7 levels were normalized to thedensitometry readings from a non-specific band detected by the secondaryIgG. Five eyes were used for each time point, except for the PARP levelsfor day 5 after detachment for which only four eyes were used. Allstatistical comparisons were performed using a paired t-test.

The cleaved, or active form of caspase 3 was elevated in the detachedretinas as compared to the attached retinas. The level ofcleaved-caspase 3 increased as a function of time after detachment, witha peak at approximately three days (see, FIG. 1). No cleaved-caspase 3was detected in the control eye or in the attached portion of the retinain the experimental eye.

The ratio of the active to inactive form of caspase 9 also increased asa function of time after creation of the experimental retinal detachment(see, FIG. 2). The peak level of cleaved-caspase 9 was seen at three tofive days after creation of the detachment. The caspase 7 antibody wasable only to detect the pro-form of the protein. There was, however, asignificant difference in the amount of the pro-form detected in theprotein extract from the detached retinas as compared to the attachedretinas (see, FIG. 3). Western blotting with antibodies against PARP (acomponent of the apoptosis cascade downstream of caspase 7) detected anincrease in the level of cleaved-PARP that was maximal at five daysafter detachment (see, FIG. 4). P-values for the comparisons betweendetached and attached retinas are shown in FIGS. 1-4.

The results demonstrate that caspase 3, caspase 7 and caspase 9 are allactivated in photoreceptor cells following retinal attachment.

Example 2: Preservation of Photoreceptor Viability Following RetinalDetachment

The type of experiment provided herein may show that the viability ofphotoreceptor cells in a detached region of a retina can be maintainedby administering a caspase inhibitor to an affected eye.

Retinal detachments are surgically induced in Brown-Norway rats asdiscussed in Example 1. The caspase inhibitor,Z-Val-Ala-Asp-fluoromethylketone is dissolved in dimethyl sulfoxide(DMSO) to give the final concentrations of 0.2 mM, 2 mM, and 20 mM.After creating the retinotomy with the subretinal injector, a smallamount of Healon® sodium hyaluronate is injected in the subretinal spaceso as to elevate the retina. After retinal elevation, a Hamilton syringewith a 33 gauge needle is introduced through the retinotomy site, and 25μl of inhibitor is injected into the region of detachment. About 25minutes later, Healon® sodium hyaluronate is injected, via the sameretinotomy site, to maintain the retinal detachment. Healon® sodiumhyaluronate is injected until resistance is detected.

Only the right eyes of rats are used in evaluating the role of theZ-VAD-FMK inhibitor. The left eyes serve as the controls. Five animalsare used for each concentration of inhibitor (namely, no inhibitor, 0.2mM inhibitor, 2 mM inhibitor, and 20 mM inhibitor). For the no inhibitorcontrol, 25 μl of DMSO is injected into the region of detachmentfollowed by Healon® sodium hyaluronate.

After 72 hours, the eyes are enucleated and the rats euthanized. Theenucleated eyes are paraffin embedded as described in Example 1. Then, 6μm sections from the posterior segments are analyzed by TUNEL stainingas described in Example 1. It is contemplated that there will be fewerphotoreceptor cells in the region of retinal detachment that stainTUNEL-positive in eyes treated with the caspase inhibitor relative toeyes that have not been treated with the caspase inhibitor.

Example 3: Detection of mRNA Levels of bFGF, TNF-Alpha, IL-1 Beta, andMCP-1

This example demonstrates that mRNA levels of bFGF, TNF-alpha, IL-1beta, and MCP-1 increase in retinal samples following retinaldetachment.

Two groups of eyes from six adult male Brown Norway rats (300-450 g,Charles River, Boston, Mass.) were examined. One group of eyes was thecontrol group having non-detached retinas, while the other group of eyeswas the experimental group having detached retinas (each rat had onecontrol and one experimental eye). Rats were anesthetized with a 50:50mixture of ketamine (100 mg/mL) and xylazine (20 mg/mL) (both fromPhoenix Pharmaceutical, St. Joseph, Mo.). Pupils were dilated with atopically applied mixture of phenylephrine (5.0%) and tropicamide(0.8%). A sclerotomy was created approximately 2 mm posterior to thelimbus with a 20-gauge needle. Care was taken not to damage the lensduring creation of the sclerotomy. A Glaser subretinal injector(20-gauge shaft with a 32-gauge tip, BD Biosciences, Franklin Lakes,N.J.) connected to a syringe filled with 10 mg/mL Healon® sodiumhyaluronate (Pharmacia and Upjohn Co., Kalamazoo, Mich.) was thenintroduced into the vitreous cavity. A retinotomy was created in theperipheral retina with the tip of the subretinal injector, and thesodium hyaluronate was slowly injected into the subretinal space. Thus,one half of the retina was detached. Retinal detachments were createdonly in the right eye of each animal, with the left eye serving as thecontrol. Three days after retinal detachment, the rats were sacrificedwith an overdose of sodium pentobarbital and the eyes were enucleated.The neural retina was dissected in a cold pool of PBS and frozenimmediately with powdered dry ice.

Total RNA was extracted with a Micro-to-Midi™ Total RNA PurificationSystem (Invitrogen, Carlsbad, Calif.) according to the manufacture'sinstructions. Each retina was homogenized manually with 600 μul of RNAlysis solution, and the same volume of 70% ethanol was added. Themixture was applied to an RNA spin cartridge and centrifuged at 12,000×gfor 15 seconds at 25° C. The cartridge was washed with wash buffer I andtwice with wash buffer II. Total RNA was eluted with 30 μl of RNase-freewater. To decrease the contamination of genomic DNA, DNase I(Invitrogen) was added followed by a 15 minute incubation at roomtemperature. The concentration of total RNA was measured with a UVspectrometer (MUTO, JAPAN). Three micrograms of total RNA was used in areverse transcription reaction using the SuperScript III First-StrandSynthesis System for RT-PCR (Invitrogen). First-strand cDNA wasamplified using an ABI7700 real-time PCR system with a SYBR® green PCRcore kit (Applied Biosystems) and PCR primer sets as listed in Table 1,in which the forward primers (F1) appear in the 5′ to 3′ direction fromleft to right and the reverse primers (R1) appear in the 3′ to 5′direction from left to right.

PCR amplification was performed for 40 cycles with two step PCR methods.Denaturation was at 96° C. for 30 seconds and annealing and extensionwere at 60° C. for 90 seconds. The quality of PCR products was evaluatedby agarose gel electrophoresis and staining with ethidium bromide. Forrelative comparison of each gene, the Ct value of real-time PCR data wasanalyzed with the 2(-Delta Delta C(T)) method (Livak et al. (2001)METHODS. 25: 402-8). To standardize the amount of sample cDNA added toeach reaction, the Ct value of each target gene was subtracted by the Ctvalue of endogenous control (18rRNA).

The data from the experimental and control groups were analyzed using aT-test and StatView 4.11J software for the Macintosh computer (Abacusconcepts Inc., Berkeley, Calif.). The significance level was set atP<0.05 (*). All values are expressed as the mean±standard deviation(SD). The bar chart shown in FIG. 6 indicates the expressional change ofeach mRNA examined by real-time PCR of the six rats 3 days followingretinal detachment. The Y axis presents the relative value of theexperimental right eye versus the untreated left control eye. bFGF,TNF-alpha, IL-1 beta, and MCP-1 were significantly increased 3 daysafter retinal detachment.

Additionally, for each type of mRNA of interest (bFGF, PEDF, TNF-alpha,IL-1 beta, HGF, CNTF, TGF beta, and IGF 1), the amount of mRNA from eachof the six eyes of a group was averaged. Then, the average amount of atype of mRNA from the detached group was compared with the amount of atype of mRNA from the non-detached group. The results are shown in FIG.5. As can be seen, the level of bFGF is approximately 11 times greaterin the detached group than in the non-detached group; the level ofTNF-alpha is approximately 8 times greater in the detached group than inthe non-detached group; and the level of IL-1 beta is approximately 3 to4 times greater in the detached group than in the non-detached group.

TABLE 1 Primer Set for real-time PCR. 18rRNAF1-CAGTGAAACTGCGAATGGCTCATT (SEQ ID NO: 7)R1-CCCGTCGGCATGTATTAGCTCTAGA (SEQ ID NO: 8) bFGFF1-TCTTCCTGCGCATCCATCCAGA (SEQ ID NO: 9)R1-CAGTGCCACATACCAACTGGAG (SEQ ID NO: 10) BDNFF1-CTTGGACAGAGCCAGCGGATTTGT (SEQ ID NO: 11)R1-CCGTGGACGTTTGCTTCTTTCATG (SEQ ID NO: 12) NT-3F1-TCTGCCACGATCTTACAGGTGAACA (SEQ ID NO: 13)R1-CGCCTGGATCAACTTGATAATGAGG (SEQ ID NO: 14) NT-4F1-TACCCTGGCAAGAGAGACGAGGAA (SEQ ID NO: 15)R1-CCACCGTGCATGGTTTATGATACG (SEQ ID NO: 16) GDNFF1-TGCCCTTCGCGCTGACCAGTGACA (SEQ ID NO: 17)R1-TTCGAGGAAGTGCCGCCGCTTGTT (SEQ ID NO: 18) IGF-1F1-TTCAGTTCGTGTGTGGACCAAGG (SEQ ID NO: 19)R1-GCTTCAGCGGAGCACAGTACATCT (SEQ ID NO: 20) HGFF1-AGATGAGTGTGCCAACAGGTGCAT (SEQ ID NO: 21)R1-AGGTCAAATTCATGGCCAAACCC (SEQ ID NO: 22) PDGFAF1-CACTGTTAAGCATGTGCCGGAGAA (SEQ ID NO: 23)R1-CCAGATCAAGAAGTTGGCCGATGT (SEQ ID NO: 24) PDGFBF1-CTTGAACATGACCCGAGCACATTCT (SEQ ID NO: 25)R1-ATCGATGAGGTTCCGCGAGATCT (SEQ ID NO: 26) TNF-alphaF1-CCCAGACCCTCACACTCAGATCAT (SEQ ID NO: 27)R1-GCAGCCTTGTCCCTTGAAGAGAA (SEQ ID NO: 28) IL-1 betaF1-TCAGGAAGGCAGTGTCACTCATTG (SEQ ID NO: 29)R1-ACACACTAGCAGGTCGTCATCATC (SEQ ID NO: 30) TGEbeta2F1-AATGGCTCTCCTTCGACGTGACA (SEQ ID NO: 31)R1-CCTCCAGCTCTTGGCTCTTATTTGG (SEQ ID NO: 32) CNTFF1-GCCGTTCTATCTGGCTAGCAAGGA (SEQ ID NO: 33)R1-GCCTCAGTCATCTCACTCCAACGA (SEQ ID NO: 34) MCP-1F1-ATGCAGGTCTCTGTCACGCTTCTG (SEQ ID NO: 35)R1-GACACCTGCTGCTGGTGATTCTCTT (SEQ ID NO: 36) VEGFF1-TCTTCCAGGAGTACCCCGATGAGA (SEQ ID NO: 37)R1-GGTTTGATCCGCATGATCTGCAT (SEQ ID NO: 38) Angiopoietin-1F1-GCCCAGATACAACAGAATGCGGTT (SEQ ID NO: 39)R1-CTCCAGCAGTTGGATTTCAAGACG (SEQ ID NO: 40) Angiopoietin-2F1-CTCGGAAACTGACTGATGTGGAAGC (SEQ ID NO: 41)R1-TGTCCTCCATGTCCAGCACTTTCTT (SEQ ID NO: 42) CTGFF1-ACCCAACTATGATGCGAGCCAACT (SEQ ID NO: 43)R1-AATTTTAGGCGTCCGGATGCACT (SEQ ID NO: 44) PEDFF2-GCTGTTTCCAACTTCGGCTACGAT (SEQ ID NO: 45)R2-AGAGAGCCCGGTGAATGACAGACT (SEQ ID NO: 46)

Example 4: Characterization of Cytokine Response in Experimental RetinalDetachment

This example characterizes the molecular and cellular responses thatoccur after retinal detachment by quantifying growth factors, cytokines,and chemokines in a rat model of experimental retinal detachment.

Initial experiments characterized cytokine, chemokine, and growth factorresponses to retinal detachment by determining changes in geneexpression in the whole neural retina following retinal detachment using19 different PCR primer sets. An identified subset of cytokines andgrowth factors was further investigated to determine the cellular originand time course of gene expression by combining laser capturemicrodissection (LCM) with quantitative real-time PCR (QPCR), and byimmunohistochemistry. LCM allows capture of specific cells in ahistological section using laser irradiation (see, for example, Sgroi etal. (1999) CANCER RES. 59: 5656-61). Real-time PCR detects small changesin gene expression using PCR amplification and quantification of mRNAlevels with the 2(-Delta Delta C(T)) method (Livak et al. (2001)METHODS. 25: 402-8). In order to pinpoint cellular sources of cytokineproduction, LCM and QPCR techniques were combined to isolate cells fromvarious retinal layers and quantify the mRNA in those layers. Data fromthese techniques demonstrates a relationship between retinal detachment,cytokine activation and photoreceptor cell death. Additionally, forcertain cytokines and growth factors, protein levels after retinaldetachment were examined.

Methods

Retinal detachment was induced by subretinal injection of sodiumhyaluronate. Retinal tissues were collected at various time points (1,3, 6, 24, 72 hours) after inducing retinal detachment. Neural retina washomogenized, and mRNA expression was quantified by QPCR. To identify thecellular sources of expressed genes, samples from various retinal layerswere obtained using LCM. Immunohistochemistry and EnzymeLinked-Immuno-Sorbent Assay (ELISA) were performed to show expressionalchanges of proteins. TUNEL staining was used in order to assessphotoreceptor death induced by retinal detachment with or withoutsubretinal administration of cytokines.

Retinal Detachment Procedure

Fifty-five adult male Brown Norway rats (200-300 g, Charles River,Boston, Mass.) were used in this study. Rats were anesthetized with a1:1 mixture of ketamine (100 mg/mL) and xylazine (20 mg/mL; both fromPhoenix Pharmaceutical, St. Joseph, Mo.). Pupils were dilated with atopically applied mixture of phenylephrine (5.0%) and tropicamide (0.8%;Massachusetts Eye and Ear Infirmary internal formulary preparation). Asclerotomy was created approximately 2 mm posterior to the limbus with a22-gauge needle. Care was taken not to damage the lens during creationof the sclerotomy. A Glaser subretinal injector (20-gauge shaft with a32-gauge tip, BD Biosciences, San Jose, Calif.) connected to a syringefilled with 10 mg/mL Healon® sodium hyaluronate (Pharmacia and UpjohnCo., Kalamazoo, Mich.) was then introduced into the vitreous cavity. Aretinotomy was created in the peripheral retina with the tip of thesubretinal injector, and the sodium hyaluronate was slowly injected intothe subretinal space, causing detachment of one half of the retina.

Retinal detachments were created only in the right eye of each animal,with the left eye serving as a control. At specified days after retinaldetachment, rats were sacrificed with an overdose of sodiumpentobarbital, and the eyes were enucleated. Each neural retina wasimmersed and dissected in cooled PBS and was frozen immediately with dryice. Samples were kept at −80° C. until used in further experiments.

RNA Extraction and RT-PGR Examination

Total RNA was extracted with a Micro-to-Midi™ Total RNA PurificationSystem (Invitrogen Corp., Carlsbad, Calif.) according to themanufacturer's instructions. Each retina was homogenized manually with600 μl of RNA lysis solution and added to an equivalent volume of 70%ethanol. The mixture was applied to an RNA spin cartridge andcentrifuged at 12,000×g for 15 seconds at 25° C. The cartridge waswashed once with wash buffer I and twice with wash buffer II. Total RNAwas eluted using 30 μl of RNase-free water. To prevent contamination ofgenomic DNA, DNase I (Invitrogen Corp., Carlsbad, Calif.) was added,followed by a 15 minute incubation at room temperature. Total RNAconcentration was measured using a UV spectrophotometer (UV-1201,Shimadzu corp., Kyoto, JAPAN). Three micrograms of total RNA was used ina reverse transcription reaction using the SuperScript™ III First-StrandSynthesis System for RT-PCR (Invitrogen, Carlsbad, Calif.). First-strandcDNA was amplified using an ABI7700 real-time PCR system with a SYBR®green PCR core kit (Applied Biosystems, Foster City, Calif.) and the PCRprimer sets listed in Table 1.

PCR amplification was performed for 40 cycles with two-step PCR methods.Denaturation was at 96° C. for 30 seconds and annealing and extensionwere at 60° C. for 90 seconds. The quality of the PCR products wasevaluated by agarose gel electrophoresis and staining with ethidiumbromide. For relative comparison of each gene, the Ct value of real-timePCR data was analyzed with the 2(-Delta Delta C(T)) method (Livak et al.(2001) METHODS. 25: 402-8). To normalize the amount of sample cDNA addedto each reaction, the Ct value of each target gene was subtracted by theCt value of endogenous control (18rRNA).

Laser Capture Microdissection (LCM)

Three days after retinal detachment, eyes were enucleated and embeddedin Tissue-Tek° OCT™ compound (Sakura Finetechnical Co., Ltd., Tokyo,Japan). Transverse cryosections measuring 12 microns and including theoptic nerve were made with a cryostat (Micron, Germany) and mounted onSuperfrost® Plus slides (Fisherbrand, Pittsburgh, Pa.). Before LCM,sections were dehydrated using 75% ethanol, DEPC water twice, 75%ethanol, 95% ethanol, and 100% ethanol for one minute each and Xylen for5 minutes.

Tissue separation with LCM was achieved using a laser at 70 mW laserpower for 0.75 seconds with a spot size of 7.5 μm for the GCL and forthe RPE. These settings were changed to 90 mW for 1.2 seconds with a 15μm spot size for the INL and for the ONL. LCM was performed on eachnuclear layer (GCL, INL, ONL, RPE) from 16 sections in the area of thedetached retina and from the corresponding area of the undetached, lefteye. Samples collected from cell layers of the left, intact eye servedas controls. After collection of aimed cells with the LCM caps(Arcturus, Mountain View, Calif.), total RNA was extracted with aPicoPure RNA Isolation Kit (Arcturus, cat# KIT0202) according to themanufacturer's instructions with the recommended optional DNasetreatment (Qiagen, catalog#79254, Valencia, Calif.). Total RNA waseluted with 30 μl of Elution Buffer. Subsequently, 24 μl of the solutioncontaining total RNA was used for QPCR.

ELISA of TNF-Alpha, IL-1 Beta, and MCP-1

Samples of neural retina were collected at 6 and 72 hours after retinaldetachment. For each retina, protein was extracted with 200 μl of RIPAlysis buffer (50 mM Tris [pH 8.0], 1% NP-40, 0.5% sodium deoxycholate,0.1% SDS, 150 mM NaCl) containing one tablet of protease inhibitorcocktail (Complete, Roche Diagnostics, Alameda, Calif.) and wassonicated at 10 watts using a Branson Sonifier® 250 (Branson UltrasonicsCorp., Danbury, Conn.) for 2 seconds. A 30 minute incubation on icefollowed. The supernatant was collected after centrifugation at 14,000×g(Micromax RF, Thermo IEC, Needham Heights, Mass.) for 30 minutes at 4°C., and the total protein concentration was measured with a DC proteinassay kit (Bio-Rad, Hercules, Calif.). One hundred micrograms of totalprotein were used for ELISA (Biosource, Camarillo, Calif.), and ELISAwas performed according to the protocol that was provided with the kit.The absorbance at 450 nm wavelength was measured using a 96-wellplate-reading spectrophotometer (Spectramax 190, Molecular Devices,Sunnyvale Calif.).

Immunohistochemisny

Isolated retinas were fixed in 4% paraformaldehyde (PFA) at 4° C.overnight and then cryoprotected with PBS (0.1 M phosphate buffer [pH,7.4], 0.15 M NaCl) containing 20% sucrose. Retinal specimens were frozenin Tissue-Tek® OCT™ compound, and 10 μm sections were prepared with acryostat to include the optic nerve. Sections were mounted ontoSuperfrost® slides, placed in blocking buffer (PBS containing 10% goatserum, 0.5% gelatin, 3% BSA, and 0.2% Tween20), and incubated withrabbit anti-rat polyclonal bFGF (Santa Cruz Biotech. Inc., Santa Cruz,Calif., 1:200), rabbit anti-rat polyclonal TNF-alpha (1:200), rabbitanti-rat polyclonal IL-1 beta (Pierce Biotechnology, Inc., Rockford,Ill., 1:200) or rabbit anti-rat polyclonal MCP-1 (Peprotec, Rocky Hill,N.J., 1:200). For double staining, mouse monoclonal antibody againstglial fibrillary acidic protein (GFAP, Sigma-Aldrich, 1:400), as amarker of astrocytes, or mouse monoclonal antibody against glutaminesynthetase (BD Biosciences, San Jose, Calif., 1:200), as a marker ofMüller cells, were used. The same procedure was used for the negativecontrol but without the primary antibody. Sections were then incubatedwith fluorescence-conjugated secondary antibody, either goat anti-mouseimmunoglobulin G (IgG) conjugated to Alexa Fluor 488 (green color) oranti-rabbit IgG conjugated to Alexa Fluor 546 (red color) (MolecularProbes, Eugene, Oreg.). Sections were mounted with Vectashield mountingmedia with propidium iodide (Vector Laboratories, Burlingame, Calif.).Photomicrographs of retinal sections were taken 2 mm from the center ofthe optic nerve head using fluorescent microscopy (DMRXA camera, Leica,Germany) and OpenLab software, version 2.2.5 (Improvision Inc.,Lexington, Mass.).

TUNEL Staining

Subretinal administration of select cytokines was performed followingthe creation of retinal detachment. Twenty four hours after retinaldetachment and subretinal administration of 5 μl of PBS, rat recombinantTNF-alpha (0.1 μg/μ1), rat recombinant IL-1 beta (0.1 μg/μl), or ratrecombinant MCP-1 (0.1 μg/μl), the eyes were harvested, fixed overnightwith 4% PFA, and cryoprotected with 20% sucrose. TUNEL staining wasperformed using the ApopTag® Fluorescein In Situ Apoptosis detection kit(S7110, Chemicon International, Inc., Temecula, Calif.). The center ofthe retinal detachment lesion was photographed. TUNEL-positive cellswere counted in a masked fashion, and standard error was determined.

Statistics

Data from the experimental and control groups were analyzed with anunpaired T-test using StatView 4.11J software for a Macintosh computer(Abacus concepts Inc., Berkeley, Calif.). The significance level was setto P<0.05 (*). Except where otherwise noted, values were expressed asmean±standard deviation (SD).

Results

Significant increases in bFGF, TNF-alpha, IL-1 beta, and MCP-1 mRNA wereobserved in neural retina 72 hours after retinal detachment. LCMrevealed increased expression of mRNA for bFGF and MCP-1 in all retinallayers, with bFGF especially evident in the ONL and MCP-1 evident in theINL. TNF-alpha mRNA was significantly increased in ONL and INL. IL-1beta mRNA was significantly increased in GCL.

Time course experiments showed that bFGF mRNA was increased after 24hours and that MCP-1 mRNA was detectable after 1 hour. However, thecurve of time dependent increase for MCP-1 mRNA was similar to that ofbFGF mRNA. TNF-alpha and IL-1 beta mRNA were increased within 1 hourfollowing retinal detachment; however, TNF-alpha mRNA showed a secondincrease after 6 hours. ELISA analysis revealed that TNF-alpha, IL-1beta, and MCP-1 proteins were increased significantly at 6 hours afterretinal detachment. Immunohistochemistry indicated bFGF and TNF-alphaprotein expression in the whole retina, while IL-1 beta protein wasspecifically expressed in the astrocytes and MCP-1 protein was expressedin the Müller cells. Subretinal administration of exogenous MCP-1protein increased TUNEL-positive cells in ONL 24 hours after retinaldetachment.

Gene-Expression Following Retinal Detachment

Retinal detachment was induced in the rats, and the expression of 19different genes was examined by QPCR at 72 hours following detachment.The nineteen genes that were examined included bFGF; brain-derivedneurotrophic factor (BDNF); neurotrophin-3,4 (NT-3,4); glial cellline-derived neurotrophic factor (GDNF); insulin growth factor-1(IGF-1); hepatocyte growth factor (HGF); platelet-derived growth factorA,B (PDGFA,B); TNF-alpha; IL-1 beta; transforming growth factor beta 2(TGF-beta2); ciliary neurotrophic factor (CNTF); MCP-1; vascularendothelial growth factor (VEGF); angiopoietin-1,2 (Angio-1,2);connective tissue growth factor (CTGF): and PEDF.

FIG. 7, which displays certain data also shown in FIG. 6, indicates theexpression pattern of these nineteen genes and shows the average foldincrease in expression of each gene's mRNA in detached retinas of righteyes (OD) as compared to the expression of the same gene's mRNA inundetached retinas of left eyes (OS), for six rats. The genes are shownon the x-axis and the average fold increase is shown on the y-axis. Theaverage fold increase in mRNA expression of bFGF (11.6±6.0, p<0.0001),TNF-alpha (5.7±3.3, p=0.0015), IL-1 beta (3.8±2.5, p=0.0003), and MCP-1(149.3±53.3, p<0.0001) was significant at 72 hours following retinaldetachment. Other cytokines and growth factors showed no significantchange in the average fold increase in expression in the detachedretinas in right eyes (OD) versus the non-detached retinas in left eyes(OS).

LCM Analysis

QPCR analysis was performed on samples collected from various retinallayers using LCM. The RPE contains retinal pigment epithelial cells; theONL primarily contains photoreceptors; the INL is composed of multiplecell types including amacrine cells, bipolar cells, horizontal cells,and Miller cells, whose cellular processes span the retina; and the GCLprimarily contains retinal ganglion cells (RGC), displaced amacrinecells, and, to some extent, astrocytes and endothelial cells.

FIG. 8 shows results of QPCR analysis on LCM-collected samples of threeto five rats. The average fold increase in mRNA expression of bFGF wassignificantly increased in all layers. The average fold increase in mRNAexpression of TNF-alpha was significantly decreased in the RPE (0.2±0.1,p=0.0185), and increased in the ONL (15.6±12.8, p=0.0211) and in the INL(54.4±20.9, p=0.0008), but not significantly changed in the GCL. Theaverage fold increase in mRNA expression of IL-1 beta was significantlyincreased only in the GCL (9.4±3.5, p=0.0467), but not in the otherlayers. The average fold increase in mRNA expression of MCP-1 wasincreased significantly in ONL (18.6±8.0, p=0.0493), INL (187.0±67.1,p=0.0104), and GCL (4.0±1.2, p=0.0164), but unchanged in RPE. These datasuggest that the distribution of genes induced after retinal detachmentis specific to various retinal layers. The highest level of geneinduction was, for bFGF, in the ONL, for TNF-alpha and MCP-1 in the INL,and for IL-1 beta in the GCL.

Time Course Evaluation Following Retinal Detachment

Three days following retinal detachment, significant average foldincreases in mRNA expression were detected for bFGF, TNF-alpha, IL-1beta, and MCP-1, as shown in FIG. 7. To see earlier time points forthese responses, retinal tissues were harvested at 1 h, 3 h, 6 h, and 24h after induction of detachment. FIGS. 9A-D show the time courseevaluation of the average fold increase in mRNA expression in totalretina with detachment in right eyes (OD) as compared to total retinawithout detachment in left eyes (OS) at 1 hour (FIG. 9A), 3 hours (FIG.9B), 6 hours (FIG. 9C), and 24 hours (FIG. 9D).

As shown in FIG. 9A, at one hour after retinal detachment the averagefold increases in mRNA expression of TNF-alpha, IL-1 beta, and MCP-1were significant. However, MCP-1 mRNA levels were not as elevated at 1hour compared to 72 hours following retinal detachment (as shown in FIG.7). The average fold increase of mRNA expression of bFGF was not changedat this time point.

As shown in FIG. 9B, at three hours after retinal detachment the averagefold increases of mRNA expression of IL-1 beta and MCP-1 weresignificant, but the fold increase of IL-1 beta was lower than after 1hour. The average fold increases of mRNA expression of TNF-alpha andbFGF were not changed significantly.

As shown in FIG. 9C, at six hours after retinal detachment the averagefold increase of mRNA expression of TNF-alpha was at its peak level, butthe average fold increases of mRNA expression of bFGF, IL-1 beta, andMCP-1 were not significantly changed from their 3 hour levels.

As shown in FIG. 9D, at twenty-four hours after retinal detachment theaverage fold increase of mRNA expression of bFGF increasedsignificantly. The average fold increases in mRNA expression ofTNF-alpha, IL-1 beta, and MCP-1 remained significant.

These data indicate that gene expressional changes for TNF-alpha, IL-1beta, and MCP-1 can be detected as early as one hour following retinaldetachment, and that increased expression of bFGF becomes significant by24 hours after retinal detachment. As FIG. 9E shows, the peak of IL-1beta induction was 1 hour after retinal detachment. After 3 hours IL-1beta dropped and then remained constant. TNF-alpha showed relativelyheightened levels at 1 hour, dropped at 3 hours, and peaked at 6 hours(FIGS. 9A-C). As shown in FIG. 9E, the curve of MCP-1 induction wassimilar to that of bFGF, however MCP-1 increased significantly within 1hour. These data suggest that inducible factors, MCP-1 and bFGF, overlap24 hours later and that there may be a functional redundancy betweenthese genes.

ELISA of TNF-Alpha, IL-1 Beta, and MCP-1

To investigate changes in cytokine expression at the protein level,ELISA was performed on neural retina at 6 and 72 hours after retinaldetachment. Total protein levels in detached retinas versus undetachedretinas did not vary significantly, as shown in Table 2. However, theratios of TNF-alpha, IL-1 beta, and MCP-1 proteins in the right eye (OD,with detached retina) as compared to the left eye (OS, with non-detachedretina) were significantly increased at six hours. Seventy-two hoursafter retinal detachment, the significant increase in MCP-1 expressionwas sustained. FIGS. 10A and 10B show the quantitative results ofTNF-alpha, IL-1 beta, and MCP-1 protein expression as assessed by ELISA.In FIG. 10A, the y-axis represents measured cytokine levels (mean±SD) ineyes with detached retinas (RD+) and in eyes with undetached retinas(RD−) with statistical significance determined using the Mann-Whitney Utest. FIG. 10B shows and alternate view of the same data, in which they-axis shows the ratio of the average ELISA result from detached retinalsamples (OD) versus control retinal samples (OS) at 6 and 72 hours afterdetachment for 4 rats (n=4, mean±SD). At six hours after retinaldetachment in the untreated control left eye, the average concentrationof TNF-alpha was 1.24 pg/μg total protein (n=4), IL-1 beta was 1.12pg/μg total protein, and MCP-1 was 0.87 pg/μg total protein, while inthe right eye with retinal detachment, TNF-alpha was 1.57 pg/μg totalprotein, IL-1 beta was 1.41 pg/μg total protein, and MCP-1 was 1.35pg/μg total protein. These data indicate that 6 hours after retinaldetachment the relative protein levels of TNF-alpha, IL-1 beta, andMCP-1 were significantly increased.

TABLE 2 Total protein per retina of detached retina and control. RD RD(−) RD6 h (μg/retina) 921.5 ± 22.2 929.0 ± 46.9 N.S RD72 h(μg/retina)960.3 ± 83.5 950.3 ± 38.4 N.S

Immunohistochemistry of bFGF, TNF-Alpha, IL-1 Beta and MCP-1 afterRetinal Detachment

The distribution of bFGF, TNF-alpha, IL-1 beta, and MCP-1 proteins wasanalyzed at 6 and 72 hours after retinal detachment byimmunohistochemistry using polyclonal antibodies against these proteins.In untreated retinal sections, immunoreactivity of bFGF and TNF-alphawas weakly detectable in the entire retina; immunoreactivity of IL-1beta was distributed in the vitreal surfaces of the GCL; and MCP-1 wasslightly detectable in the cell bodies in the GCL and INL.

Six hours following retinal detachment, immunoreactivity of bFGF wasunchanged. But after 72 hours, the immunoreactivity of bFGF wasincreased in the ONL, INL, and GCL, and especially in the vitrealsurfaces of the GCL and in the middle row of the INL (Miller cells).Monocytes localized on the outer segment of photoreceptors, whichappeared at 72 hours after retinal detachment, also showedimmunoreactivity for bFGF.

TNF-alpha was increased in the ONL and INL at 6 hours after retinaldetachment, but, by 72 hours, the intensity of the signal was decreased.The infiltrating monocytes showed TNF-alpha immunoreactivity at 72 hoursafter retinal detachment.

IL-1 beta immunoreactivity was significantly increased in the GCL 6hours and 72 hours after retinal detachment. However, the signal after 6hours was stronger than at 72 hours, and monocytes in the subretinalspace were not stained. Double labeling with antibody against GFAP, anastrocyte marker, demonstrated that the IL-1 beta immunoreactivityco-localized with astrocytes.

Immunoreactivity for MCP-1 was significantly increased in the INL, withthe appearance of spindle-shaped cells that are indicative of Müllercells, by 6 hours. Greater staining was observed at 72 hours. Thesubretinal monocytes also expressed the MCP-1 protein. The expression ofMCP-1 in the INL was co-localized with glutamine synthetase, a Müllercell marker.

These data suggested that bFGF and TNF-alpha were increased in the wholeretina, while IL-1 beta and MCP-1 were specifically increased inastrocyte or Müller cells, respectively, in the neural retina.

TUNEL Staining 24 Hours after Retinal Detachment with SubretinalAdministration of Cytokines

To investigate effects of cytokines on photoreceptor cell death inducedby retinal detachment, TUNEL staining was performed 24 hours afterretinal detachment with subretinal administration of PBS, TNF-alpha (0.1μg/μ1), IL-1 beta (0.1 μg/μl), or MCP-1 (0.1 μg/μl). FIG. 11A shows theaverage cell TUNEL-positive cells counted per field, with error barsrepresenting standard error. In the control condition with PBS,TUNEL-positive cells were detected in the ONL 24 hours after retinaldetachment (15.3±5.4 cells/field, n=7). The number of TUNEL-positivecells did not change significantly with subretinal administration ofTNF-alpha and IL-1 beta. However, subretinal administration of MCP-1increased the number of TUNEL-positive cells in the ONL 24 hours afterretinal detachment (67.7±46.5 cells/field, p=0.0138, n=7). FIG. 11Bshows an alternate view of the data shown in FIG. 11A, with the y-axisshowing the average cell TUNEL-positive cells counted per squaremillimeter, with error bars representing standard error. These datasuggest that MCP-1 enhances photoreceptor cell death induced by retinaldetachment.

Discussion

Experiments described in this example characterized the expressionalgene changes of growth factors, cytokines, and chemokines in an animalmodel of retinal detachment. These experiments showed that mRNAs ofTNF-alpha, IL-1 beta, and MCP-1 were detected very early after retinaldetachment, and bFGF began to increase at 24 hours. By 72 hoursfollowing retinal detachment, bFGF, TNF-alpha, IL-1 beta, and MCP-1 weresignificantly increased. Examination of the distribution of these genesafter retinal detachment using QPCR with samples collected by LCM showedthat mRNA of bFGF was most increased in the photoreceptor layer,although the induction was also detected significantly in all othernuclear layers (RPE, INL, GCL). TNF-alpha was increased in the ONL andthe INL, and decreased in the RPE. IL-1 beta was specifically increasedin the GCL, and MCP-1 was increased in the ONL and the INL.

LCM sampling of cells from the various layers of neural retinacorrelated with the distribution of bFGF, TNF-alpha, IL-1 beta, andMCP-1 detected by immunohistochemistry. Astrocytes, Müller cells, andsubretinal monocytes produced bFGF, TNF-alpha, IL-1 beta, and/or MCP-1in the retina. Specifically, the immunoreactivity of bFGF and TNF-alphawas increased over the entire neural retina, while IL-1 beta and MCP-1were increased in the astrocytes or Müller cells, respectively.Subretinal administration of MCP-1 with retinal detachment significantlyenhanced the number of TUNEL-positive cells in the ONL 24 hours afterretinal detachment, demonstrating that MCP-1 enhances photoreceptorretinal detachment-induced cell death.

The other cytokines and growth factors assessed in the experimentsdescribed in this example showed no significant change in the averagefold increase in expression of mRNA in the detached retinas in the righteyes (OD) versus the non-detached retinas in the left eyes (OS).Regulation of gene expression of bFGF, TNF-alpha, IL-1 beta, and MCP-1offers therapeutic avenues to treat retinal detachment and preventphotoreceptor loss, retinal gliosis, and proliferative changes, all ofwhich cause significant vision loss in patients.

Example 5: Neuroprotective Effect of TNF-Alpha Suppression FollowingRetinal Detachment

This example confirms that administration of agents that suppressTNF-alpha, in this case goat TNF-alpha blocking antibody and etanercept,protects against cell death following retinal detachment.

Retinal detachments were experimentally induced in the right eye ofAdult Male Norway Brown rats, using the retinal detachment proceduredescribed in Example 4. Retinal detachments were created only in theright eye of each animal, and the left eye served as a control.Following retinal detachment, various treatments were administeredsubretinally using a Hamilton syringe equipped with a 32-gauge needle.The tip of needle was introduced through a sclerotomy and retinal holeinto the subretinal space, and 5 μl of solution, either normal goatserum (0.1 mg/mL), goat anti-TNF-alpha antibody (0.1 mg/mL), oretanercept (2 mg/mL), was injected over 3 minutes. Seventy-two hoursafter retinal detachment, the eyes were subjected to TUNEL analysis.

FIGS. 12 and 13 show TUNEL-positive responses in the ONL at 72 hoursfollowing retinal detachment. As shown in FIG. 12, animals treated withetanercept following retinal detachment had a significantly decreasedTUNEL-positive response in the ONL at 72 hours as compared to controls.Similarly, FIG. 13 shows that animals treated with goat anti-TNF-alphaantibody (TNFa) following retinal detachment had a significantlydecreased TUNEL-positive response in the ONL at 72 hours followingretinal detachment as compared to animals treated with normal goat serum(NGS) or controls left untreated (RD72 hours). These data indicate thatadministration of agents that suppress TNF-alpha, such as goat TNF-alphablocking antibody or etanercept, protects against photoreceptor celldeath following retinal detachment.

Example 6: TNF-Alpha, TNF Receptor, and MCP-1 Deficient Animals ShowLess Apoptotic Cell Death Following Retinal Detachment

This example shows that TNF-alpha deficient mice (TNF−/−), TNF Receptors1A and 1B double deficient mice (TNFR−/−), and MCP-1 deficient miceexhibit less apoptotic cell death following retinal detachment ascompared to wild-type mice.

Knock-out mice deficient in TNF-alpha, TNF Receptors 1A and 1B, or MCP-1were anesthetized by intraperitoneal injection of a ketamine (62.5mg/kg) and xylazine (12.5 mg/kg) mixture. For each animal, afterdilation of the animal's pupil with 1% cyclopentolate and 2.5%phenylephrine hydrochloride, a scleral puncture was made at thesupemasal equator using a glass micropipette. One microliter of vitreousfluid was removed to reduce ocular pressure. Then, a glass micropipettewas introduced into the subretinal space, and one microliter of Healon®GV sodium hyaluronate (Pharmacia & Upjohn, Uppsala, Sweden) was injectedinto the subretinal space. Mice receiving scleral punctures served as acontrol. At 72 hours after retinal detachment, the eyes were subjectedto TUNEL analysis.

As shown in FIG. 14, mice deficient in MCP-1 (CCL2) showed lessapoptosis than wild-type (wild) mice at 72 hours following retinaldetachment. Additionally, as shown in FIG. 15, mice deficient inTNF-alpha (TNF KO) and mice deficient in TNF Receptors 1A and 1B (TNFRKO) showed less apoptosis than wild-type (Wild) mice at 72 hoursfollowing retinal detachment. These data further link TNF-alpha, TNFReceptors 1A and 1B, and MCP-1 with apoptosis of photoreceptors andvalidate them as appropriate treatment targets.

Example 7: Neuroprotective Effect of MCP-1 Suppression Following RetinalDetachment

This example contemplates that administration of agents that suppressMCP-1 protects against cell death following retinal detachment.

Retinal detachments are experimentally induced in the right eye of AdultMale Norway Brown rats, using the retinal detachment procedure describedin Example 4. Retinal detachments are created only in the right eye ofeach animal, and the left eye serves as a control. Following retinaldetachment, various treatments are administered subretinally using aHamilton syringe equipped with a 32-gauge needle. The tip of needle isintroduced through a sclerotomy and retinal hole into the subretinalspace, and 5 μl of solution containing a suitable concentration of aMCP-1 suppressing agent is injected over 3 minutes.

It is contemplated that treatment with an MCP-1 suppressing agent,including any of those listed herein, following retinal detachment willshow decreased TUNEL-positive responses in detached retinas at 72 hoursas compared to controls. This indicates that administration of agentsthat suppress MCP-1 protects against photoreceptor cell death followingretinal detachment.

Example 8: Neuroprotective Effect of IL-1 beta Suppression FollowingRetinal Detachment

This example contemplates that administration of agents that suppressIL-1 beta protects against cell death following retinal detachment.

Retinal detachments are experimentally induced in the right eye of AdultMale Norway Brown rats, using the retinal detachment procedure describedin Example 4. Retinal detachments are created only in the right eye ofeach animal, and the left eye serves as a control. Following retinaldetachment, various treatments are administered subretinally using aHamilton syringe equipped with a 32-gauge needle. The tip of needle isintroduced through a sclerotomy and retinal hole into the subretinalspace, and 5 μl of solution containing a suitable concentration of anIL-1 beta suppressing agent is injected over 3 minutes.

It is contemplated that treatment with an IL-1 beta suppressing agent,including any of those listed herein, following retinal detachment willshow decreased TUNEL-positive responses in detached retinas at 72 hoursas compared to controls. This indicates that administration of agentsthat suppress IL-1 beta protects against photoreceptor cell deathfollowing retinal detachment.

Example 9: Neuroprotective Effect of bFGF Induction Following RetinalDetachment

This example contemplates that administration of agents that induce bFGFprotect against cell death following retinal detachment.

Retinal detachments are experimentally induced in the right eye of AdultMale Norway Brown rats, using the retinal detachment procedure describedin Example 4. Retinal detachments are created only in the right eye ofeach animal, and the left eye serves as a control. Following retinaldetachment, various treatments are administered subretinally using aHamilton syringe equipped with a 32-gauge needle. The tip of needle isintroduced through a sclerotomy and retinal hole into the subretinalspace, and 5 μl of solution containing a suitable concentration of abFGF inducing agent is injected over 3 minutes.

It is contemplated that treatment with an bFGF inducing agent, includingany of those listed herein, following retinal detachment will showdecreased TUNEL-positive responses in detached retinas at 72 hours ascompared to controls. This indicates that administration of agents thatinduce bFGF protects against photoreceptor cell death following retinaldetachment.

Example 10: Intravitreal Administration of PEDF

This example confirms that an intravitreal administration of PEDF has nosignificant effect on the mRNA expression of bFGF, TNF-alpha, or IL-1beta, and fails to significantly protect against photoreceptor celldeath following retinal detachment.

In these experiments, retinal detachments were created in Brown Norwayrats by injecting 10% hyaluronic acid into the subretinal space using atransvitreous approach. Treatment with PEDF (BioProducts MD, Middletown,Md.) or control vehicle was administered immediately after detachment.In an initial set of experiments, the treatment groups included:intravitreal administration of 2.5 μg PEDF (n=6); intravitrealadministration of 5.0 μg PEDF (n=9); subretinal administration of 2.5 μgPEDF (n=6); subretinal administration of 5.0 μg PEDF (n=9); intravitrealadministration of control vehicle (n=3); and subretinal administrationof control vehicle (n=3). In a second set of experiments, the treatmentgroups included: intravitreal administration of 5.0 μg PEDF (n=15);subretinal administration of 5.0 μg PEDF (n=15); intravitrealadministration of control vehicle (n=9); and subretinal administrationof control vehicle (n=9).

For both sets of experiments, all eyes were enucleated 72 hours afterretinal detachment and embedded in paraffin. Four micron sectionsthrough the area of detachment, including the optic nerve, wereobtained. Light microscopy was performed at 3× and at 32× magnificationusing hematoxylin-eosin staining. TUNEL staining was performed using acommercial kit (Oncogene, San Diego, Calif.) and viewed at 20×magnification using green fluorescein staining for TUNEL and blue DAPInuclear staining. TUNEL-positive cells were counted per millimeter oftissue by a masked observer. The mean number of TUNEL-positive cells permillimeter of tissue was determined by averaging the number ofTUNEL-positive cells per mm of tissue in three sections of each eye.Additionally, a realtime polymerase chain reaction (PCR) was performedfor a variety of cytokines, including bFGF, TNF-alpha, and IL-1 beta, onextracted retina from normal eyes (n=6), untreated eyes with retinaldetachment (n=6), PEDF-treated eyes with retinal detachment (n=6) andPBS-treated eyes with retinal detachment (n=6).

Light microscopic analysis of detached retinas showed the presence ofpyknotic nuclei in the outer nuclear layer, disruption of the normalorganization of the photoreceptor outer segments, and loss ofphotoreceptor nuclei. In the initial set of experiments, TUNEL-stainingof detached retina from rats treated with a subretinal injection ofcontrol vehicle showed multiple TUNEL-positive cells in the ONL, whereasTUNEL-staining of detached retina from rats treated with a subretinalinjection of 5.0 μg PEDF showed fewer TUNEL-positive cells in the ONL.Quantitatively, as shown in Table 3, the initial set of experimentsshowed that the mean number of TUNEL-positive cells per millimeter oftissue was greater in control eyes with detached retinas having asubretinal sham injection (mean SD, 63.8±11.9) versus eyes with detachedretinas that were subretinally injected with 2.5 μs PEDF (17.6±15.5) or5.0 μs PEDF (30.4±18.1). These differences were statisticallysignificant by a two-tailed t-test (p=0.007 and p=0.016, respectively).There was no statistically significant difference in the number ofTUNEL-positive cells found in the intravitreally-injected control eyesversus the intravitreally-injected PEDF eyes.

TABLE 3 Number of TUNEL-Positive Cells in Initial Set of Experiments.Treatment Mean SD P Intravitreal Control 23.3 11.0 Intravitreal PEDF 2.5μg 16.9 14.3 0.100 Intravitreal PEDF 5.0 μg 28.4 15.7 0.190 SubretinalControl 63.8 11.9 Subretinal PEDF 2.5 μg 17.7 15.5 0.007 Subretinal PEDF5.0 μg 30.4 18.1 0.016

However, as shown in Table 4, the second set of experiments conductedwith larger numbers of sample and control eyes showed no statisticaldifference between control eyes with detached retinas having asubretinal sham injection (mean±SD, 38.6±32.2) and eyes with detachedretinas that were subretinally injected with 5.0 μs PEDF (35.5±42.1). Aswith the initial set of experiments, the second set of experimentsshowed no statistically significant difference in the number ofTUNEL-positive cells found in the intravitreally-injected control eyesversus intravitreally-injected PEDF eyes.

TABLE 4 Number of TUNEL-Positive Cells in Second Set of Experiments.Treatment Mean SD P Intravitreal Control 30.2 22.0 Intravitreal PEDF 5.0μg 30.3 28.5 0.122 Subretinal Control 38.6 32.2 Subretinal PEDF 5.0 μg35.5 42.1 0.847

A realtime PCR was performed for a variety of cytokines, including bFGF,PEDF, TNF-alpha, IL-1 beta, HGF, CNTF, TGF beta, and IGF 1. The amountof each mRNA from each of six eyes treated with a 5.0 μg intravitrealinjection of PEDF just after detachment was averaged. That average wascompared with the average amount of mRNA from each of six eyes treatedwith the PBS control vehicle just after detachment. The results for theintravitreal injection are shown in FIG. 16. As can be seen,intravitreal administration of PEDF does not significantly alter themRNA levels of bFGF, TNF-alpha, IL-1 beta, or any of the cytokinesmeasured.

Overall, the experiments described in this example show that in a largersample set, administration of PEDF, either subretinally orintravitreally, has no significant effect on photoreceptor cell deathfollowing retinal detachment as measured using TUNEL.

INCORPORATION BY REFERENCE

The entire content of each patent and non-patent document disclosedherein is expressly incorporated herein by reference for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A method of preserving the viability ofphotoreceptor cells disposed within a retina of a mammalian eyefollowing retinal detachment, the method comprising: administering to amammal having an eye in which a region of the retina has been detachedan amount of a neuroprotective agent selected from the group consistingof a substance capable of suppressing endogenous MCP-1, an MCP-1antagonist, and combinations thereof sufficient to preserve theviability of photoreceptor cells disposed within the region of thedetached retina.
 2. The method of claim 1, wherein the neuroprotectiveagent is administered to the mammal prior to reattachment of the regionof detached retina.
 3. The method of claim 1, wherein theneuroprotective agent is administered to the mammal after reattachmentof the region of detached retina.
 4. The method of claim 1, wherein theneuroprotective agent is administered locally or systemically. 5-6.(canceled)
 7. The method of claim 4, wherein at least oneneuroprotective agent is administered by intraocular, intravitreal, ortranscleral administration.
 8. The method of claim 1, wherein theneuroprotective agent reduces the number of photoreceptor cells in theregion that die following retinal detachment.
 9. The method of claim 1,wherein the retinal detachment occurs as a result of a retinal tear,retinoblastoma, melanoma, diabetic retinopathy, uveitis, choroidalneovascularization, retinal ischemia, pathologic myopia, or trauma.10-18. (canceled)
 19. A method of preserving the viability ofphotoreceptor cells disposed within a retina of a mammalian eyefollowing retinal detachment, the method comprising: administering to amammal having an eye in which a region of the retina has been detachedan amount of a neuroprotective agent selected from the group consistingof a substance capable of suppressing endogenous IL-1 beta, an IL-1 betaantagonist, and combinations thereof sufficient to preserve theviability of photoreceptor cells disposed within the region of thedetached retina.
 20. The method of claim 19, wherein the neuroprotectiveagent is administered to the mammal prior to reattachment of the regionof detached retina.
 21. The method of claim 19 or 20, wherein theneuroprotective agent is administered to the mammal after reattachmentof the region of detached retina.
 22. The method of claim 19, whereinthe neuroprotective agent is administered locally or systemically.23-24. (canceled)
 25. The method of claim 22 or 21, wherein at least oneneuroprotective agent is administered by intraocular, intravitreal, ortranscleral administration.
 26. The method of claim 19, wherein theneuroprotective agent reduces the number of photoreceptor cells in theregion that die following retinal detachment.
 27. The method of claim19, wherein the retinal detachment occurs as a result of a retinal tear,retinoblastoma, melanoma, diabetic retinopathy, uveitis, choroidalneovascularization, retinal ischemia, pathologic myopia, or trauma. 28.A method of preserving the viability of photoreceptor cells disposedwithin a retina of a mammalian eye following retinal detachment, themethod comprising: administering to a mammal having an eye in which aregion of the retina has been detached an amount of a neuroprotectiveagent selected from the group consisting of a substance capable ofinducing endogenous bFGF, a bFGF mimetic, and combinations thereofsufficient to preserve the viability of photoreceptor cells disposedwithin the region of the detached retina.
 29. The method of claim 28,wherein the neuroprotective agent is administered to the mammal prior toreattachment of the region of detached retina or after reattachment ofthe region of detached retina.
 30. (canceled)
 31. The method of claim28, wherein the neuroprotective agent is administered locally orsystemically. 32-33. (canceled)
 34. The method of claim 31, wherein atleast one neuroprotective agent is administered by intraocular,intravitreal, or transcleral administration.
 35. The method of claim 28,wherein the neuroprotective agent reduces the number of photoreceptorcells in the region that die following retinal detachment.
 36. Themethod of claim 28, wherein the retinal detachment occurs as a result ofa retinal tear, retinoblastoma, melanoma, diabetic retinopathy, uveitis,choroidal neovascularization, retinal ischemia, pathologic myopia, ortrauma.